Treatment of diseases and conditions mediated by increased phosphorylation

ABSTRACT

The invention provides methods for the treatment of diseases and conditions mediated by increased phosphorylation, such as inflammation and cancer. The invention also provides methods for the inhibition of increased phosphorylation in cells, tissues and organs. The methods utilize a phosphate acceptor compound (PAC). The invention also provides products comprising a PAC.

This invention claims the benefit of priority under 35 U.S.C. §119(e)from U.S. Provisional Application 60/429,924, filed Nov. 27, 2002, whichis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to methods for the treatment of diseases andconditions mediated by increased phosphorylation, such as inflammationand cancer. The invention also relates to methods for the inhibition ofincreased phosphorylation in cells, tissues and organs. The methodsutilize a phosphate acceptor compound (PAC). The invention also relatesto products comprising a PAC.

BACKGROUND OF THE INVENTION

Signal transduction is the cascade of processes by which anextracellular signal interacts with a receptor at the cell surface toultimately effect a change in cell functioning. Protein phosphorylationplays a key role in signal transduction. Protein phosphorylation isperformed by protein kinases, and virtually all aspects of cellfunctioning in one way or another depend on kinase activity. Notsurprisingly, then, abnormal (usually increased) kinase activity hasbeen related to a host of diseases and disorders.

Protein kinases catalyze the transfer of phosphate from adenosinetriphosphate (ATP) to specific amino acid residues (almost always aserine, threonine or tyrosine residue) of proteins. Several features ofkinases make them ideally suited to function in signal transduction. Oneis that they often have overlapping target substrate specificities whichallows “cross-talk” among different signaling pathways, allowing for theintegration of different signals. A second feature is that the kinasesare organized into several modular functional domains. These domainsappear to have been mixed and matched through evolution to produce thelarge protein kinase family, and the kinases are structurally andcatalytically similar. A third feature is their speed. The kinetics ofphosphorylation and dephosphorylation are extremely rapid, providing forrapid responses and short recovery times, which in turn makes repeatedsignal transmission possible.

Given their roles in numerous diseases and in signal transduction,considerable effort has been made to develop protein kinase inhibitors.As far as is known, all of this effort has been directed at developingspecific inhibitors that inhibit only a single kinase. However, theeffort to find specific inhibitors of single kinases is a daunting task.Recent estimates predict the presence of greater than 2,000 proteinkinase genes in the human genome. Shanley, Crit. Care Med., 30 (No. 1,Suppl):S80-S88 (2002); Cohen, Current Opinion in Chemical Biology,3:459-465 (1999). Also, the very features described above which makekinases so useful in signal transduction, and which have made themevolve to become central to almost every cellular function, also makethem extremely difficult to study and understand. U.S. Pat. No.6,383,790. Unfortunately, the enormous number of kinases and thesimilarities between them have frustrated the discovery and design ofspecific inhibitors. U.S. Pat. No. 6,383,790. Further, because thekinase networks are highly degenerate and interconnected in unknownways, there is considerable uncertainty with regard to which kinasesshould be targeted for inhibition to treat many diseases. U.S. Pat. No.6,383,790. Moreover, it is by no means clear that a specific inhibitorof a given kinase will have any effect on a given disease. U.S. Pat. No.6,383,790. Since kinases can be highly promiscuous, there is asignificant chance that inhibiting one kinase will simply force anotherkinase to take its place. U.S. Pat. No. 6,383,790.

From the foregoing it is clear that it would be desirable to havemethods and products for reducing unwanted phosphorylation without theneed to identify specific inhibitors for individual kinases. It wouldfurther be desirable to have methods and products for the effectivetreatment of diseases and conditions mediated by increasedphosphorylation without the need to identify specific inhibitors forindividual kinases. The invention provides such methods and products.

SUMMARY OF THE INVENTION

In particular, the invention provides methods and products for thetreatment of diseases and conditions mediated by increasedphosphorylation. The invention also provides methods and products forinhibiting increased phosphorylation in cells, tissues and organs. Themethods and products of the invention utilize a phosphate acceptorcompound (PAC). A PAC is a compound that is capable of beingphosphorylated. Phosphorylation of PACs has the effect of reducing theamount of ATP and phosphate groups available for phosphorylation bykinases of their normal substrates, thereby inhibiting the unwantedincreased phosphorylation. Thus, the identity of the PACs is notcrucial, and it is not necessary to know which specific kinases areinvolved in causing the unwanted increased phosphorylation.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: A bar graph showing the amount interleukin 8 (IL-8) versustreatment.

DETAILED DESCRIPTION OF THE PRESENTLY-PREFERRED EMBODIMENTS OF THEINVENTION

As described above, the invention provides methods and products for thetreatment of diseases and conditions mediated by increasedphosphorylation. The invention also provides methods and products forinhibiting increased phosphorylation in cells, tissues and organs. Themethods and products utilize a phosphate acceptor compound (PAC).

“Treat” and variations thereof are used herein to mean to cure, prevent,eliminate, ameliorate, alleviate or reduce the severity of a disease orcondition, or of at least some of the symptoms or effects thereof.

As used herein a “disease or condition mediated by increasedphosphorylation” means a disease or condition caused by, exacerbated byor involving increased phosphorylation of proteins and/or peptides ofthe animal suffering from the disease or condition.

“Increased phosphorylation” means a level of phosphorylation above thenormal level found in the absence of such a disease or condition.Increased phosphorylation is caused by a net increase in kinase activitywhich, in turn, is caused by increased kinase activity, decreasedphosphatase activity, or both.

“Inhibit” and variations thereof are used herein to mean reduce,eliminate or prevent.

A. Phosphate Acceptor Compounds

As used herein, a “phosphate-acceptor compound” or “PAC” is a compoundthat is capable of being phosphorylated. PACs may be extracellular PACs(“EPACs”) or intracellular PACs (“IPACs”).

Phosphorylation of PACs has the effect of reducing the amount of ATP andphosphate groups available for phosphorylation by kinases of theirnormal substrates, thereby inhibiting unwanted increasedphosphorylation. Thus, the identity of the PACs is not crucial, and itis not necessary to know which kinase(s) are involved in causing theunwanted increased phosphorylation.

In cases where it is known or suspected that a particular kinase isresponsible for, or at least involved in causing, the unwanted increasedphosphorylation, one or more substrates of the kinase, if known, can beused as the PAC(s). The use of such substrates may more efficiently orcompletely inhibit the unwanted increased phosphorylation, but the useof such substrates is not necessary. Further, the use of such substratesmay not result in any improvement in inhibition since, as noted above,kinases can be highly promiscuous, and other kinases may simply assistthe kinase suspected of being responsible for, or involved in causing,the unwanted increased phosphorylation in performing its function.

1. Extracellular Phosphate Acceptor Compounds

By “extracellular” is meant that the extracellular phosphate acceptorcompound (EPAC) does not penetrate sufficiently inside cells to interactwith intracellular pathways involving phosphorylation. Doing so couldseriously damage or even kill the cells. The EPAC may remain entirelyoutside the cell, may interact with or bind to receptors and/or othermolecules found on or in cell membranes, or may even penetrate thecellular membrane, as long as the EPAC does not penetrate it in such amanner that it can interact with intracellular pathways involvingphosphorylation.

The EPACs are substrates for phosphorylation by membrane-bound and/orcirculating/soluble kinases. EPACs compete with the normal substrates tobe phosphorylated by kinases, and phosphorylation of the EPACs reducesor prevents phosphorylation of extracellular and intracellular proteins,peptides and other compounds (phosphorylation of the EPACs has theeffect of reducing the amount of ATP and phosphate groups available forphosphorylation of both extracellular and intracellular proteins,peptides and other compounds). Also, phosphorylation of the EPACs bymembrane-bound kinases reduces or prevents signal transduction from theoutside to the inside of cells.

In addition, phosphorylated proteins and peptides can bind metal ions,including iron and copper ions. As a result, they can function asantioxidants. Thus, the EPAC proteins and peptides of the inventionshould provide an added antioxidant benefit after they arephosphorylated.

Suitable EPACs include proteins and peptides that comprise one or morephosphorylation sites. As noted above, virtually all protein kinasesphosphorylate serine, threonine and/or tyrosine residues, and over 1000phosphorylation sites comprising serine, threonine and/or tyrosineresidues are now known. See, e.g., Kreegipuu et al., “PhosphoBase, adatabase of phosphorylation sites: release 2.0,” Nucleic Acids Res.,27(1):237-239 (1999) and http://www.cbs.dtu.dk/databases/PhosphoBase/.The proteins and peptides may be naturally-occurring proteins andpeptides, fragments of such proteins and peptides and synthetic proteinsand peptides (including mutated and partially synthetic forms ofnaturally-occurring proteins and peptides and wholly synthetic proteinsand peptides) that comprise at least one phosphorylatable amino acid.The proteins and peptides must be of a sufficient size (typicallygreater than about 20 amino acids in length) and/or have a charge (i.e.,be hydrophilic) that causes them to remain extracellular.

At least one of the phosphorylation sites of an EPAC must, of course, beunphosphorylated for it to function as a phosphate acceptor.Unphosphorylated proteins and peptides can be produced by chemicalsynthesis using methods well known in the art. For instance, theproteins and peptides can be synthesized by standard solid-phase peptidesynthesis methods. Suitable techniques are well known in the art, andinclude those described in Merrifield, in Chem. Polypeptides, pp. 335-61(Katsoyannis and Panayotis eds. 1973); Merrifield, J. Am. Chem. Soc.,85, 2149 (1963); Davis et al., Biochem. Int'l, 10, 394-414 (1985);Stewart and Young, Solid Phase Peptide Synthesis (1969); U.S. Pat. Nos.3,941,763 and 5,786,335; Finn et al., in The Proteins, 3rd ed., vol. 2,pp. 105-253 (1976); and Erickson et al. in The Proteins, 3rd ed., vol.2, pp. 257-527 (1976). Proteins and peptides made by chemical synthesismay be comprised of L-amino acids, D-amino acids, or combinationsthereof, except that the phosphorylatable amino acids (serines,threonines and/or tyrosines) are preferably not D-amino acids. The useof proteins and peptides composed of one or more D-amino acids isdesirable because proteins and peptides containing D-amino acids areresistant to proteolytic enzymes, such as those that would beencountered upon administration of the proteins and peptides to ananimal or that would be present in an excised organ perfused with asolution containing the proteins or peptides. Also, the use of D-aminoacids should not alter the ability of the proteins or peptides to bephosphorylated.

Unphosphorylated proteins and peptides can also be produced in bacteriaby recombinant DNA techniques. See U.S. Pat. No. 5,942,254.Phosphorylated proteins and peptides can be produced in other hosts andthen dephosphorylated as described below. Recombinant DNA techniques,vectors and reagents for expressing proteins in bacteria and other hostsare well known in the art. See, e.g., Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (1982), Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.(1989).

Some naturally-occurring unphosphorylated proteins and peptides that canfunction as EPACs are known. For instance, α_(S1)-casein found in humancolostrum is substantially unphosphorylated.

Proteins and peptides which are phosphorylated must be at leastpartially dephosphorylated prior to use. As used herein “at leastpartially dephosphorylated” means that the number of phosphorylatedamino acids present in the population of proteins or peptides is reducedby at least about 10%. The number of phosphorylated amino acids presentin the population of proteins or peptides is preferably reduced by atleast about 35%, more preferably by at least about 50%, even morepreferably by at least about 70%, most preferably by at least about 90%,prior to use. Also, as used herein, those proteins and peptides producedor found unphosphorylated or substantially unphosphorylated come withinthe definition of proteins and peptides that are “at least partiallydephosphylated.”

Methods of dephosphorylating proteins and peptides that arephosphorylated are well known. In particular, proteins and peptides canbe dephosphorylated enzymatically or chemically. See, e.g., U.S. Pat.Nos. 6,355,297 and 5,068,118, Miller et al., J. Biol. Chem.,257:6818-6824 (1982), Jiang and Mine, J. Agric. Food Chem., 48:990-994(2000), and Example 2. For enzymatic dephosphorylation, any of a largenumber of phosphatases can be employed. Preferred is an acid phosphataseor an alkaline phosphatase. These phosphatases are non-specificphosphomonoesterases and can be used to dephosphorylate most proteinsand peptides. Preferred is an alkaline phosphatase, most preferably fromEscherichia coli. Also, serine/threonine phosphatases, tyrosinephosphatases, tyrosine/threonine phosphatases and combinations of theforegoing could be used. Suitable phosphatases are availablecommercially from numerous companies, including Sigma-Aldrich Co.,Worthington Biochemical Corp., Takara Bio Inc., CHIMERx, and Promega. Inchemical dephosphorylation, the phosphates are cleaved off by heatingthe proteins and peptides (preferably at 35° C.-70° C.) at alkaline pH(preferably about pH 10-12) for a time sufficient to obtain the desiredamount of dephosphorylation. The presently preferred conditions aregiven in Example 2 and Jiang and Mine, J. Agric. Food Chem., 48:990-994(2000).

The extent of phosphorylation of the protein or peptide before and afterdephosphorylation can be determined. Suitable methods are well known inthe art. See, e.g., Miller et al., J. Biol. Chem., 257:6818-6824 (1982)and Example 2. Also, kits are available commercially that can be used tomeasure phosphorylation levels. Such kits are available from, e.g.,Pierce (Phosphoprotein Phosphate Estimation Kit based on the alkalinehydrolysis of phosphate from serine and threonine residues followed byquantification of the phosphate by the use of Malachite Green andammonium molybdate). In addition, an immunoassay using appropriateantibodies (e.g., antibodies specific for phosphorylated serine,threonine and tyrosine residues) could be used. Such antibodies areavailable commercially from, e.g., Zymed Laboratories.

Specific proteins useful in the practice of the invention includephosvitins and fragments thereof. Phosvitins are egg yolk proteins.Chicken phosvitin has been reported to have 109 phosphorylation sites(Miller et al., J. Biol. Chem., 257:6818-6824 (1982)), and isparticularly preferred for use herein. Chicken phosvitin can be preparedby methods well known in the art (see, e.g., Japanese application JP3056500) and is available commercially from, e.g., U.S. BiochemicalAmersham and Sigma-Aldrich. It can be dephosphorylated as describedabove.

Other suitable proteins for use in the practice of the invention includecaseins. Whole casein, α-casein, one of the several isoforms ofα-casein, β-casein, γ-casein and/or κ-casein and/or fragments of any ofthe foregoing can be used. Caseins have multiple phosphorylation sites.For instance, β-casein has five phosphorylation sites. Whole caseins,α-caseins, isoforms of α casein, β-caseins, γ-caseins, and κ-caseins areavailable commercially (from, e.g., Sigma-Aldrich) or can made beprepared by methods well known in the art (see, e.g., U.S. Pat. Nos.5,068,118, 5,739,407, 5,795,611, 5,942,274 and 6,232,094 andhttp://wvvw.worthington-biochem.com/CASA). Preferred are caseins made byrecombinant DNA techniques in bacteria, since they will not bephosphorylated. Also, as noted above, α_(S1) casein from human colostrumis naturally unphosphorylated and is, therefore, very convenient to use.It is available commercially from Sigma-Aldrich. Dephosphorylation ofcaseins, if necessary, can be performed as described above. See, e.g,U.S. Pat. Nos. 6,355,297 and 5,068,118.

Other suitable EPACs for use in the practice of the invention includeblood proteins and peptides. If the proteins and/or peptidesadministered to an animal are from the same species of animal as the onebeing treated, the proteins and/or peptides should not be immunogenic.Thus, homologous blood proteins and peptides are particularly suitablefor systemic administration.

One or a mixture of blood proteins and/or peptides can be used as EPACs.For instance, all of the proteins and peptides in a pooled plasma orpooled serum sample could be dephosphorylated and used in the practiceof the invention. Alternatively, individual plasma proteins or peptideshaving one or more phosphorylation sites could be isolated from plasmaor serum or could be produced by recombinant DNA techniques and used inthe practice of the invention.

It has been found that human albumin can be phosphorylated. However,acetylation of the albumin was necessary for phosphorylation to takeplace. It is believed that it is the serine at position 202 that isphosphorylated and the lysine at position 199 that is acetylated. It isalso believed that, in albumin which is not acetylated, the positivelycharged side chain of the lysine interacts with the negatively chargedserine, blocking phosphorylation of the serine. When the lysine isacetylated, its charge is neutralized and it no longer interacts withthe serine, and the serine can be phosphorylated. Acetylated humanalbumin has also been found to be present in plasma and serum,especially in individuals who are taking aspirin, and it could beisolated from pooled plasma or serum, dephosphorylated, and used in thepractice of the present invention. Alternatively, albumin could beproduced by recombinant DNA techniques (preferably in bacteria so itwill not be phosphorylated) and acetylated. Methods of acetylatingproteins are well known. For instance, albumin can be acetylated byincubating plasma (containing esterases) with 1 mM aspirin for 1 hour at37° C., and the acetylated albumin isolated from the plasma by methodswell known in the art. Also, serum or albumin (preferably albuminproduced by recombinant DNA techniques) can be incubated with aspirin inthe presence of an esterase or with N-acetyl aspartate in the presenceof an acylase to acetylate the albumin. Albumin could also be acetylatedusing acetic anhydride. Of course, other acylations, such asmethylation, could be performed instead of acetylation.

Also, a peptide comprising the albumin sequence from position 199 toposition 202 can be prepared and acetylated. The sequence from position199 to position 202 of albumin is Lys Cys Ala Ser [SEQ ID NO:1]. Otheramino acids could be substituted for the cysteine and alanine residuesbetween the lysine and serine residues, and the serine residue could bereplaced by a threonine or tyrosine residue. Also, additional aminoacids could be added to either end. Also the number of amino acidsbetween the serine and lysine residues can be varied from 1-3, and thesequence repeated one or more times. Thus, another peptide useful in thepractice of the invention is Lys Ala Ser Ser Ala Lys [SEQ ID NO:2],wherein both lysine residues are acetylated. In the case of EPACs, theamino acids substituted for the cysteine and/or alanine and/or added toeither end should include sufficient charged amino acids so that thepeptides will be hydrophilic and/or add enough amino acids so that thepeptides will be at least 20 amino acids long. In particular, suchpeptides preferably will have the following sequences:

-   -   (Xaa₁)_(m)[Ac-Lys (Xaa₁)_(n)Xaa₂]_(p)(Xaa₁)_(m) or    -   (Xaa₁)_(m)[Ac-Lys (Xaa₁)_(n)Xaa₂ Xaa₂ (Xaa₁)_(n)Lys-Ac]_(p)        (Xaa₁)_(m)

wherein:

-   -   Ac is an acyl group;    -   Xaa₁ is any amino acid;    -   Xaa₂ is serine, threonine or tyrosine;    -   m is 0-10;    -   n is 1-3;    -   p is 1-5; and    -   m, n and p are selected so that the total number of amino acids        is at least about twenty and/or each Xaa₁ is selected so that        the peptide will be hydrophilic.

Additional suitable EPACs for use in the practice of the inventioninclude known kinase substrates. Many such kinase substrates are known,and some are available commercially from, e.g., Sigma-Aldrich andPromega. As noted above, the PACs act nonspecifically to reduce theamount of available ATP and phosphate groups, and these substrates maybe used as EPACs regardless of the identity of the kinase(s) responsiblefor, or involved in causing, the unwanted increased phosphorylation.

Other suitable EPACs for use in the practice of the invention includesynthetic proteins and peptides comprising at least one phosphorylatableamino acid. For instance, a protein or peptide comprising one or moreserine, threonine and/or tyrosine residues can be used. Serine is themost-commonly phosphorylated amino acid, so the protein or peptide couldcomprise only serines. Preferably, however, the protein or peptidecontains all three of these amino acids. Also preferably, the serine,threonine and/or tyrosine residues are spaced apart by one or more otheramino acids. As noted above, if a lysine is within 1-3 amino acids of aserine, the lysine should preferably be acylated. Since the proteins andpeptides are EPACs, they should contain at least about 20 amino acidsand/or be charged (i.e., be hydrophilic). Thus, preferably, some of theother amino acids besides the serines, threonines and/or tyrosines arecharged amino acids. One protein or peptide with one or morephosphorylatable amino acids, preferably a multiplicity of such aminoacids, could be used. Alternatively, a cocktail of peptides, eachcomprising a single phosphorylatable amino acid, could be used. As usedherein, “synthetic” means not naturally occurring.

Since kinases are highly promiscuous, it is anticipated that even randomsequences comprising one or more serine, threonine and/or tyrosineresidues will be phosphorylated. However, in a preferred embodiment, thesynthetic protein or peptide will comprise one or more knownphosphorylation sites. As noted above, over 1000 phosphorylation sitescomprising serine, threonine and/or tyrosine residues are now known.See, e.g., Kreegipuu et al., “PhosphoBase, a database of phosphorylationsites: release 2.0,” Nucleic Acids Res., 27(1):237-239 (1999) andhttp://www.cbs.dtu.dk/databases/PhosphoBase/. See also, Aitken, Mol.Biotechnol., 12:241-53 (1999). Also, methods have been developed foridentifying phosphorylatable peptides from random peptide libraries. SeeWu et al., Biochemistry, 13:14825-14833 (1994) and Songyang et al.,Curr. Biol., 4:973-982 (1994). Since the EPACs act nonspecifically, theidentity of the phosphorylation sites is not critical. When a pluralityof phosphorylation sites is used, each phosphorylation site may be thesame or different than the other phosphorylation site(s).

2. Intracellular Phosphate Acceptor Compounds

By “intracellular” is meant that the intracellular phosphate acceptorcompounds (IPACs) penetrate sufficiently inside cells to interact withintracellular pathways involving phosphorylation. Preferably, the IPACswill pass through the cellular membrane into the cytoplasm of the cell.

The IPACs are substrates for phosphorylation and, once inside the cells,the IPACs will compete with the normal substrates to be phosphorylatedby intracellular kinases. Phosphorylation of the IPACs inhibitsphosphorylation of intracellular proteins, peptides and other compoundsand will inhibit signal transduction processes inside cells. Given thecritical nature of phosphorylation for cell functioning, it is expectedthat IPACs will seriously harm or kill the cells that they enter. Thus,they should only be used to treat diseases and conditions, such ascancer, where the death of cells is desired or can be tolerated.Preferably, the IPACs will be targeted as described below so that theywill enter only the cells whose death is desired. Targeting isparticularly important when they are administered systemically.

Suitable IPACs include peptides that comprise one or morephosphorylation sites. The peptides may be naturally-occurring peptides,fragments of naturally-occurring proteins and synthetic peptides(including mutated or partially synthetic forms of naturally-occurringpeptides and protein fragments and wholly synthetic peptides thatcomprise at least one phosphorylatable amino acid). The peptides shouldpreferably be less than about 20 amino acids in length, more preferablyless than about 10 amino acids in length, most preferably less thanabout 5 amino acids in length, since shorter peptides are better able toenter cells. To enhance the ability of the peptides and fragments topenetrate cell membranes, the peptides will preferably be hydrophobicand/or comprise an arginine oligomer (see Rouhi, Chem. & Eng. News,49-50 (Jan. 15, 2001)). The arginine oligomer will preferably contain6-9 Arg residues (see Rouhi, Chem. & Eng. News, 49-50 (Jan. 15, 2001).The use of an arginine oligomer may be particularly desirable when thepeptide is to be administered topically or transdermally. The peptidesmay be synthesized and, if necessary, dephosphorylated as describedabove.

Specific suitable IPACs include peptides of the sequences:

-   -   (Xaa₁)_(m)[Ac-Lys (Xaa₁)_(n)Xaa₂]_(p) (Xaa₁)_(m) or    -   (Xaa₁)_(m)[Ac-Lys (Xaa₁)_(n)Xaa₂ Xaa₂        (Xaa₁)_(n)Lys-Ac]_(p)(Xaa₁)_(m)

wherein:

-   -   Ac is an acyl group;    -   Xaa₁ is any amino acid;    -   Xaa₂ is serine, threonine or tyrosine;    -   m is 0-10;    -   n is 1-3;    -   p is 1-5; and    -   m, n and p are selected so that the total number of amino acids        is less than about twenty and each Xaa₁ is selected so that the        peptide will be hydrophobic.

Additional suitable IPACs for use in the practice of the inventioninclude known kinase substrates able to penetrate cells. Preferred arethose which are hydrophobic and comprise less than about 20 amino acids.

Other suitable IPACs for use in the practice of the invention includesynthetic peptides comprising at least one phosphorylatable amino acid.For instance, a peptide comprising one or more serine, threonine and/ortyrosine residues can be used. Serine is the most-commonlyphosphorylated amino acid, so the peptide could comprise only serines.Preferably, however, the protein or peptide contains all three of theseamino acids. Also preferably, the serine, threonine and/or tyrosineresidues are spaced apart by one or more other amino acids. Since thepeptides are IPACs, they should preferably contain less than about 20amino acids and be hydrophobic. Thus, some of the other amino acidsbesides the serines, threonines and/or tyrosines should be unchargedamino acids. One peptide with one or more phosphorylatable amino acids,preferably a multiplicity of such amino acids, could be used.Alternatively, a cocktail of peptides, each comprising a singlephosphorylatable amino acid, could be used.

Since kinases are highly promiscuous, it is anticipated that even randomsequences comprising one or more serine, threonine and/or tyrosineresidues will be phosphorylated. However, in a preferred embodiment, thesynthetic peptide will comprise one or more known phosphorylation sites.As noted above, over 1000 phosphorylation sites comprising serine,threonine and/or tyrosine residues are now known. See, e.g., Kreegipuuet al., “PhosphoBase, a database of phosphorylation sites: release 2.0,”Nucleic Acids Res., 27(1):237-239 (1999) andhttp://www.cbs.dtu.dk/databases/PhosphoBase/. See also, Aitken, Mol.Biotechnol., 12:241-53 (1999). Also, methods have been developed foridentifying phosphorylatable peptides from random peptide libraries, SeeWu et al., Biochemistry, 13:14825-14833 (1994) and Songyang et al.,Curr. Biol., 4:973-982 (1994). Since the IPACs act nonspecifically, theidentity of the phosphorylation sites is not critical. When a pluralityof phosphorylation sites is used, each phosphorylation site may be thesame or different than the other phosphorylation site(s).

3. Targeting of Phosphate Acceptor Compounds

For administration to animals, the PACs can be targeted to a selectedcell, tissue or organ. Targeting of the PACs will concentrate theireffectiveness on the targeted cells, tissues or organs, reduce thelikelihood of deleterious side effects, and decrease the dose that willneed to be given. In the case of IPACs, targeting is highly desirablewhen they are administered systemically, and targeting will reduce orprevent unnecessary cell damage and death when other modes ofadministration are employed.

As used herein, “selected” cells, tissues and organs are those intendedto be affected by the PAC. In the case of IPACs, the “selected” cells,tissues and organs are those intended to be impaired, damaged ordestroyed by the IPAC. Selected cells, tissues and organs are alsosometimes referred to herein as target cells, tissues and organs.

Numerous methods of targeting therapeutic compounds to selected cells orto a selected tissue or organ are known in the art and can be used totarget the PACs of the invention to cells, tissues or organs. Forinstance, a PAC can be conjugated to a targeting molecule. The targetingmolecule can be a specific ligand known to be reactive with a targetcell, tissue or organ; such as receptor-specific ligands (e.g., achemokine or growth factor) or antibodies specific for an antigen on thesurface of the cell, tissue or organ. Suitable antibodies includepolyclonal antibodies, omniclonal antibodies, monoclonal antibodies,bispecific antibodies, humanized antibodies, chimeric antibodies,single-chain antibodies, Fab fragments, F(ab′)₂ fragments, fragmentsproduced by an Fab expression library, epitope-binding fragments of anyof the foregoing, and complementarity determining regions (CDRs). Thetargeting molecule can also be one of the homing molecules describedbelow.

Another possibility is the use of multivalent antibodies. See, e.g.,U.S. Pat. No. 5,861,156, the complete disclosure of which isincorporated herein by reference. For instance, a multivalent antibodythat binds to a PAC and to a binding protein reactive with a cellsurface marker of the target cell, tissue or organ could be used. Afterall three have been administered to an animal, the PAC will be bound tothe multivalent antibody which, in turn, will be bound to the bindingprotein which, in turn, will be bound to the cell surface marker on thetarget cell. Preferably, the binding protein is a monospecific bindingprotein, such as Fab and F(ab)′₂ fragments, Fab fusion proteins,single-chain Fv proteins (single-chain antibodies), single-chain Fvfusion proteins, chimeric antibody proteins (e.g., recombinant antibodyproteins derived from transfectoma cells), chimeric single-chainproteins and other single-chain fusion Fv analog proteins, such assingle-chain T-cell receptors. U.S. Pat. No. 5,861,156 reports that suchmonospecific binding proteins have a unique ability to penetrate solidtumors and to be rapidly cleared from the circulation if not localizedat a target site, making them extremely suitable for treatment oftumors. The multivalent antibody is any multivalent antibody, includingpolyclonal antibodies, monoclonal antibodies, heterobispecificantibodies, fragments of any of the foregoing, chimeric antibodies,bispecific single-chain antibodies, homodimeric IgG molecules, etc. Inanother embodiment, a cocktail of binding proteins is used, each bindingprotein being specific for a different surface marker on a target celland each labelled with a moiety (e.g., a peptide tag) recognized by themultivalent antibody to allow multi-site targeting of a target cell. Themultivalent antibody also recognizes the PAC, as well as the moiety.Thus, since each type of target cell has its own unique cell surfacemarker profile, the PAC can be targeted to the target cell with greaterspecificity and selectivity than with the use of a single bindingprotein specific for a single cell surface marker.

Proteins, peptides and other molecules that home to particular cells,tissues or organs can be identified by an in vivo panning technique. SeeU.S. Pat. Nos. 6,610,651, 5,622,699 and 6,296,832, the completedisclosures of which are incorporated herein by reference. Molecules canbe identified which home to cells, tissues or organs that are normal orthat exhibit a pathological state, such as inflammation, or topathological lesions, such as cancer, in a tissue or organ. In vivopanning comprises administering (by any route) a library of molecules toa subject, collecting an organ or tissue sample from the subject, andidentifying the homing molecules using various methods well known in theart. Typically, the selected organ or tissue will be processed using amethod such as HPLC, which can be used to obtain an enriched fraction ofmolecules having a defined range of molecular weights or polarity or thelike from a complex mixture. The enriched fraction of molecules then canbe further analyzed for the purposes of identifying cell, organ ortissue homing molecules using, for example, HPLC, mass spectrometry andgas chromatography. The libraries can be composed of naturally-occurringmolecules and/or nonnaturally-occurring molecules and are made bymethods well known in the art. See U.S. Pat. No. 6,610,651. These homingmolecules can be conjugated to a PAC so that the PAC will be targeted toa desired target cell, tissue or organ. Also, a homing protein orpeptide and a PAC could be expressed as a fusion protein usingrecombinant DNA techniques. The homing molecules can also be used todeliver genes to target cells, tissues and organs. Thus, a gene encodinga PAC (IPAC or EPAC) could be delivered into a target cell, tissue ororgan using the homing molecules, where it could be expressed.

B. Therapeutic Methods and Pharmaceutical Products

The invention provides methods of treating diseases and conditionsmediated by increased phosphorylation. An EPAC can be used to treat anysuch disease or condition. An IPAC should only be used to treat diseasesand conditions, such as cancer, where cell death is desired or can betolerated. The invention also provides pharmaceutical productscomprising PACs.

Diseases and conditions mediated by increased phosphorylation includeinflammation, inflammatory diseases and conditions, cancer, otherproliferative disorders, autoimmune diseases, allergic reactions andother immune disorders.

Inflammation is a cascade of events through which the body responds to avariety of injuries, infections and stresses. Many of the phenomenacharacteristic of inflammation are associated with intensified signaltransduction at the cellular and molecular levels. One importantcharacteristic feature of cell signaling is phosphorylation. Enzymes,proteins, peptides and other molecules are activated by phosphorylationor dephosphorylation pathways. In many instances, when an activationevent involves phosphorylation, it is followed by acounter-dephosphorylation step so that a pulse signal is transmitted asopposed to a continuous stimulus. A balance normally exists between thephosphorylases/kinases and the phosphatases. On a molecular level,inflammation can be viewed as an imbalance in favor of the kinases.Inflammation involves expression of kinases intracellularly andexternalization or activation of kinases on the outer membrane of cellsand in some cases, inhibition of phosphatases, resulting in increasedkinase activity.

The inflammatory response is critical for stress response, fending offinfections and healing wounds, but inflammation can also be damaging.Indeed, inflammation is an important component of the pathogenic processof many diseases and disorders. In addition, the presence ofinflammation in many diseases, such as cancer, is indicative of a lessfavorable prognosis. Finally, in the extreme, inflammation may result ina life-threatening systemic response if not properly treated.

As noted above, the EPACs are substrates for phosphorylation bymembrane-bound and/or circulating/soluble kinases, which are increasedin inflammation. Phosphorylation of the EPACs reduces or preventsphosphorylation of extracellular and intracellular proteins, peptidesand other compounds which, as a result of phosphorylation, would becomeparticipants in inflammatory processes. In particular, phosphorylationof the EPACs by membrane-bound kinases reduces or prevents signaltransduction from the outside to the inside of cells. For these reasons,EPACs interfere with inflammatory processes and inhibit inflammation.

Specific inflammatory diseases and conditions treatable with the EPACsof the invention include acute respiratory distress syndrome, allergies,arthritis, asthma, autoimmune diseases (e.g, multiple sclerosis),bronchitis, cancer, Crohn's disease, cystic fibrosis, emphysema,endocarditis, gastritis, infections (bacterial, viral, yeast, fungal andparasitic), inflammatory bowel disease, inflammatory skin disorders,ischemia reperfusion, multiple organ dysfunction syndrome, multipleorgan failure, nephritis, neurodegenerative diseases (e.g., Alzheimer'sdisease, amyotropic lateral sclerosis, Huntington's chorea, Parkinson'sdisease, senile dementia), pancreatitis, psoriasis, respiratory viralinfections, sepsis, shock, systemic inflammatory response syndrome,trauma, ulcerative colitis and other inflammatory diseases, conditionsand disorders.

Cancers can be treated with either EPACs and/or IPACs. As noted above,IPACs are preferably targeted so that they enter only cancer cells.Specific cancers treatable with the PACs of the invention includecarcinomas, sarcomas, brain cancers, head and neck cancers, breastcancers, ovarian cancers, prostate cancers, gastric cancers, coloncancers, pancreatic cancers, bladder cancers, thyroid cancers, hepaticcancers, lung cancers, bone cancers, skin cancers, blood cancers,lymphomas and leukemias.

The other proliferative disorders include blood vessel proliferativedisorders, mesangial cell proliferation disorders and fibroticdisorders. Blood vessel proliferative disorders include angiogenicdiseases and conditions. An angiogenic disease or condition is a diseaseor condition involving, caused by, exacerbated by, or dependent onangiogenesis. Angiogenesis is the process of new blood vessel formationin the body. Specific angiogenic diseases and conditions treatable inaccordance with the invention include neoplastic diseases (e.g., tumors(e.g., tumors of the bladder, brain, breast, cervix, colon, rectum,kidney, lung, ovary, pancreas, prostate, stomach and uterus) and tumormetastasis), benign tumors (e.g., hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyrogenic granulomas), hypertrophy (e.g.,cardiac hypertrophy induced by thyroid hormone), connective tissuedisorders (e.g., rheumatoid arthritis and atherosclerosis), psoriasis,ocular angiogenic diseases (e.g., diabetic retinopathy, retinopathy ofprematurity, macular degeneration, corneal graft rejection, neovascularglaucoma, retrolental fibroplasia, and rubeosis), cardiovasculardiseases, cerebral vascular diseases, endometriosis, polyposis, obesity,diabetes-associated diseases, hemophiliac joints, and immune disorders(e.g., chronic inflammation and autoimmunity). The neoplastic diseasesmay be treated with either EPACs and/or IPACs, as described above. Theother angiogenic diseases and conditions are treated with EPACs. TheEPACs of the invention can also be used to inhibit the vascularizationrequired for embryo implantation, thereby providing a method of birthcontrol.

Mesangial cell proliferative disorders refer to disorders brought aboutby abnormal proliferation of mesangial cells. Mesangial cellproliferative disorders include renal diseases, such asglomerulonephritis, diabetic nephropathy, malignant nephrosclerosis,thrombotic microangiopathy syndromes and glomerulopathies. Malignantmesangial cell proliferative disorders can be treated with EPACs and/orIPACs, and the remaining disorders are treated with EPACs.

Fibrotic disorders refer to the abnormal formation of extracellularmatrices. Examples of fibrotic disorders include hepatic cirrhosis,pulmonary fibrosis and atherosclerosis. Fibrotic disorders are treatedwith EPACs.

Other proliferative disorders include hyperproliferative skin disorders,such as psoriasis, skin cancer and epidermal hyperproliferation.Psoriasis is characterized by inflammation, hyperproliferation of theepidermis and decreased differentiation of cells. Skin cancer can betreated with EPACs and/or IPACs, and the remaining disorders are treatedwith EPACs.

Autoimmune diseases treatable with the EPACs of the invention includemultiple sclerosis. Other immune disorders treatable with the EPACs ofthe invention include transplant rejection.

In a preferred embodiment of the invention, the PACs will be used totreat skin diseases and conditions. Skin diseases and conditionstreatable with an EPAC of the invention include a dermatitis, eczema,keratosis, elastosis, psoriasis, infections (e.g., measles and chickenpox), an acne, burns, sunburn, allergic reactions (e.g., rashes andhives), and any other inflammatory disease or condition. Skin cancerscan be treated with either an EPAC and/or an IPAC, as described above.

In another preferred embodiment of the invention, the PACs will be usedto treat diseases and conditions of the mouth. Mouth diseases andconditions treatable with an EPAC of the invention include leukoplakia,lichen plannus, infections and other inflammatory diseases andconditions, Mouth cancers can be treated with either an EPAC and/or anIPAC, as described above. Many other disease and conditions of themouth, such as gingivits and periodontitis, will be typically be treatedby, or under the supervision of; a dentist, and the treatment of thesedisease and conditions is described below in the section on oral careproducts and methods.

In yet another preferred embodiment of the invention, the EPACs will beused to treat diseases and conditions of, or involving, the mucousmembranes. Such diseases and conditions include allergies, infectionsand inflammatory diseases and conditions.

To treat an animal suffering from a disease or condition mediated byincreased phosphorylation, an effective amount of a PAC or a combinationof PACs is administered to the animal. As noted above, EPAC(s) can beused to treat any such disease or condition. IPAC(s) should only be usedto treat diseases and conditions, such as cancer, where cell death isdesired or can be tolerated. Preferably, the animal is a mammal, such asa rabbit, goat, dog, cat, horse, bovine or human, most preferably ahuman.

Effective dosage forms, modes of administration and dosage amounts forthe various PACs may be determined empirically, and making suchdeterminations is within the skill of the art. It is understood by thoseskilled in the art that the dosage amount will vary with the particularPAC(s) employed, whether the PAC(s) is(are) being administeredprophylactically or to treat an existing disease or condition, thespecific disease or condition to be treated, the severity of the diseaseor condition, the route(s) of administration, the type(s) ofcompositions and/or devices used to administer the PAC(s), the rate ofexcretion of the PAC(s), the duration of the treatment, the identity ofany other drugs being administered to the animal, the age, size andspecies of the animal, and like factors known in the medical andveterinary arts. In general, a suitable daily dose of a PAC of thepresent invention will be that amount of the compound which is thelowest effective dose to produce a therapeutic effect. If desired, theeffective daily dose may be administered as two, three, four, five, sixor more sub-doses, administered separately at appropriate intervalsthroughout the day. Administration of the PAC(s) should be continueduntil an acceptable response is achieved.

The PAC(s) of the present invention may be administered to an animalpatient for treatment of a disease or condition mediated by increasedphosphorylation by any suitable route of administration, includingorally, nasally, rectally, vaginally, parenterally (e.g., intravenously,intraspinally, intraperitoneally, subcutaneously, or intramuscualarly),intracisternally, transdermally, transmucosally, intracranially,intracerebrally, and topically. The preferred routes of administrationare topically, orally and locally. Examples of local administrationinclude administration intracranially, into a tumor or cancerous lesion,intraocularly, into a lesion, nasally, vaginally, anally, into thelungs, into the gastrointestinal tract and into the mouth. IPACs willoften have to be administered systemically to treat cancer. As notedabove, when an IPAC is administered systemically, it is highlypreferable that it be targeted so that it enters only or preferentiallythe selected cells, tissues or organs.

While it is possible for a PAC of the present invention to beadministered alone, it is preferable to administer the PAC as apharmaceutical formulation (composition). The pharmaceuticalcompositions of the invention comprise a PAC or a combination of PACs asactive ingredient(s) in admixture with one or morepharmaceutically-acceptable carriers and, optionally, with one or moreother compounds, drugs or other materials. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the animal.Pharmaceutically-acceptable carriers are well known in the art.Regardless of the route of administration selected, the PACs of thepresent invention are formulated into pharmaceutically-acceptable dosageforms by conventional methods known to those of skill in the art. See,e.g., Remington's Pharmaceutical Sciences.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules or as asolution or a suspension in an aqueous or non-aqueous liquid, or anoil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), and the like, each containing a predeterminedamount of a compound or compounds of the present invention as an activeingredient. A compound or compounds of the present invention may also beadministered as bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient (i.e., a PAC or combination of PACs) is mixed with oneor more pharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monosterate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may be employedas fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compound(s), may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound. Formulations of thepresent invention which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

Dosage forms for the topical, transdermal or transmucosal administrationof a compound of this invention include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches, drops and inhalants.The active compound(s) may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to acompound or compound(s) of this invention, excipients, such as animaland vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound or compoundsof this invention, excipients such as lactose, talc, silicic acid,aluminum hydroxide, calcium silicates and polyamide powder or mixturesof these substances. Sprays can additionally contain customarypropellants such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

The active ingredient (i.e., a PAC or combination of PACs of theinvention) may also be delivered through the skin using conventionaltransdermal drug delivery systems, i.e., transdermal patches, whereinthe active ingredient is typically contained within a laminatedstructure that serves as a drug delivery device to be affixed to theskin. In such a structure, the active ingredient is typically containedin a layer, or “reservoir,” underlying an upper backing layer. Thelaminated device may contain a single reservoir, or it may containmultiple reservoirs. In one embodiment, the reservoir comprises apolymeric matrix of a pharmaceutically acceptable contact adhesivematerial that serves to affix the system to the skin during drugdelivery. Examples of suitable skin contact adhesive materials include,but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes,polyacrylates, polyurethanes, and the like. Alternatively, thedrug-containing reservoir and skin contact adhesive are present asseparate and distinct layers, with the adhesive underlying the reservoirwhich, in this case, may be either a polymeric matrix as describedabove, or it may be a liquid or hydrogel reservoir, or may take someother form.

The backing layer in these laminates, which serves as the upper surfaceof the device, functions as the primary structural element of thelaminated structure and provides the device with much of itsflexibility. The material selected for the backing material should beselected so that it is substantially impermeable to the activeingredient and any other materials that are present. The backing layermay be either occlusive or nonocclusive, depending on whether it isdesired that the skin become hydrated during drug delivery. The backingis preferably made of a sheet or film of a preferably flexibleelastomeric material. Examples of polymers that are suitable for thebacking layer include polyethylene, polypropylene, polyesters, and thelike.

During storage and prior to use, the laminated structure includes arelease liner. Immediately prior to use, this layer is removed from thedevice to expose the basal surface thereof, either the drug reservoir ora separate contact adhesive layer, so that the system may be affixed tothe skin. The release liner should be made from a drug/vehicleimpermeable material.

Transdermal drug delivery devices may be fabricated using conventionaltechniques, known in the art, for example by casting a fluid admixtureof adhesive, active ingredient and vehicle onto the backing layer,followed by lamination of the release liner. Similarly, the adhesivemixture may be cast onto the release liner, followed by lamination ofthe backing layer. Alternatively, the drug reservoir may be prepared inthe absence of active ingredient or excipient, and then loaded by“soaking” in a drug/vehicle mixture.

The laminated transdermal drug delivery systems may, in addition,contain a skin permeation enhancer. That is, because the inherentpermeability of the skin to some active ingredients may be too low toallow therapeutic levels of the drug to pass through a reasonably sizedarea of unbroken skin, it is necessary to coadminister a skin permeationenhancer with such drugs. Suitable enhancers are well known in the art.

The pharmaceutical compositions of the invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, propellants such as fluorocarbons or nitrogen, and/orother conventional solubilizing or dispersing agents.

Preferred formulations for topical drug delivery are ointments andcreams. Ointments are semisolid preparations which are typically basedon petrolatum or other petroleum derivatives. Creams containing theselected active agent, are, as known in the art, viscous liquid orsemisolid emulsions, either oil-in-water or water-in-oil. Cream basesare water-washable, and contain an oil phase, an emulsifier and anaqueous phase. The oil phase, also sometimes called the “internal”phase, is generally comprised of petrolatum and a fatty alcohol such ascetyl or stearyl alcohol; the aqueous phase usually, although notnecessarily, exceeds the oil phase in volume, and generally contains ahumectant. The emulsifier in a cream formulation is generally anonionic, anionic, cationic or amphoteric surfactant. The specificointment or cream base to be used, as will be appreciated by thoseskilled in the art, is one that will provide for optimum drug delivery.As with other carriers or vehicles, an ointment base should be inert,stable, nonirritating and nonsensitizing.

Formulations for buccal administration include tablets, lozenges, gelsand the like. Alternatively, buccal administration can be effected usinga transmucosal delivery system as known to those skilled in the art.

Pharmaceutical compositions of this invention suitable for parenteraladministrations comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending or thickeningagents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drug isaccomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

The PAC(s) of the invention may be administered alone or may beadministered in combination with one or more other drugs, compounds orother materials. For instance, an EPAC can be administered incombination with one or more additional anti-inflammatory compounds,including steroids, non-steroid anti-inflammatory compounds (e.g.,aspirin, ibuprofen, etc.), and those anti-inflammatory compoundsdescribed in U.S. patent application Ser. Nos. 09/678,202, 09/922,234,and 10/186,168, and PCT applications WO 01/25265, WO 02/11676 and WO02/64620, the complete disclosures of which are incorporated herein byreference.

C. Excised Cells, Tissues and Organs

The EPACs of the invention can also be used to inhibit increasedphosphorylation in a cell, tissue or organ that has been removed from ananimal. Increased phosphorylation may be found in a cell, tissue ororgan because of inflammation. To inhibit the increased phosphorylationin a tissue or an organ, the tissue or organ is contacted with asolution (e.g., by placing the tissue or organ in the solution and/or byperfusing an organ (e.g., a kidney) with the solution) containing aneffective amount of an EPAC or combination of EPACs of the invention.Effective amounts of the EPAC(s) to include in such solutions can bedetermined empirically, and doing so is within the skill in the art. Theharvested tissue or organ may subsequently be used for transplantationinto a recipient or for research purposes (e.g., using a perfused liverto screen drugs). The EPACs of the invention can be used alone or can beused in combination with other compounds, drugs or materials.

Many suitable solutions for use with tissues and organs are known inwhich an EPAC or combination of EPACs of the invention could be used.See, e.g., Hauet et al., J. Pharmacol. Exp. Ther., 297, 946-953 (2001);Hauet et al., J. Pharmacol. Exp. Ther., 292, 254-260 (2000); Dunphy etal., Am. J. Physiol., 276, H1591-H1598 (1999); Muhlbacher et al.,Transplant Proc., 31, 2069-2070 (1999); Watts et al., J. Mol. Cell.Cardiol., 31, 1653-1666 (1999); Suzer et al., Pharmacol. Res., 37,97-101 (1998); Collins et al., Kidney Int'l, 42, Suppl. 38, S-197-S-202(1992); Paller, Ren. Fail., 14, 257-260 (1992); Baron et al., J. Surg.Res., 51, 60-65 (1991); Hisatomi et al., Transplantation, 52, 754-755(1991); Belzer et al., Transplantation, 45, 673-76 (1988); U.S. Pat.Nos. 4,798,824, 4,873,230, 4,879,283, 5,514,536, and 5,710,172; and PCTapplication WO 98/35551 (the disclosures of all of the foregoing areincorporated herein by reference).

For instance, a solution for flushing and cold storage of hearts is theCelsior™ solution (available from SangStat Medical Corp., Fremont,Calif.). Celsior™ solution contains:

TABLE A Component Concentration Mannitol 60 mmol Lactobionic Acid 80mmol Glutamic Acid 20 mmol Histidine 30 mmol Calcium Chloride 0.25 mmolPotassium chloride 15 mmol Magnesium Chloride 13 mmol Sodium hydroxide100 mmol Reduced Glutathione 3 mmol Water For Injection Up to 1 liter

The accepted standard solution for preservation of kidneys is theUniversity Of Wisconsin solution (available from Barr Laboratories undertradename ViaSpan®) which has the following composition:

TABLE B Component Concentration Function Raffinose 30 mM Impermeant:suppression of (17.83 g/L) hypothermic cell swelling Lactobionic acid100 mM Impermeant: suppression of (35.83 g/L) hypothermic cell swellingPentafraction 50 g/L Colloid: reduction of inter- (hydroxyethyl starch)stitial edema and endothelial cell swelling Glutathione 3 mM Antioxidant(0.992 g/L) 1 mM Inhibition of xanthine oxidase Allopurinol (0.136 g/L)activity and purine metabolism/ reduction of oxygen free radicals 5 mMRestoration of high energy Adenosine (1.34 g/L) phosphate 25 mM pHbuffer: maintenance of intra- Potassium phosphate (3.4 g/L) cellularsodium and potassium concentrations: restoration of high energyphosphate 5 mM Preservation of intracellular Magnesium sulfate (1.23g/L) magnesium concentration 100 mM Maintenance of intracellularPotassium hydroxide (5.61 g/L) sodium and potassium concentrationsSodium hydroxide 27 mM Maintenance of intracellular sodium and potassiumconcentrations Solution is pH adjusted to 7.4 with either sodiumhydroxide or hydrochloric acid. Final: Sodium = 29 mM; Potassium = 125mM; mOsm/L = 320 ± 10 Immediately prior to use, to formulate the finalsolution, aseptically add: Penicillin G 200,000 units, regular insulin40 units, and dexamethasone 16 mg.

An EPAC or combination of EPACs of the invention could be used in eitherof these two solutions, variations of these solutions, or in one of theother numerous solutions known in the art or which will be developed.The EPAC(s) may be included in the solution or supplied separately(e.g., in lyophilized form) and added at the time of use.

Cells isolated from an animal can be stored or cultured in a mediumcontaining an effective amount of an EPAC or combination of EPACs of theinvention. Many suitable media are known. Effective amounts of the EPACsto include in the medium can be determined empirically, and doing so iswithin the skill in the art. The EPAC(s) may be included in the mediumor supplied separately (e.g., in lyophilized form) and added at the timeof use. The cells may be administered to a recipient in need thereof(e.g., for gene therapy) or may be used for research purposes.

The invention further provides a kit for inhibiting increasedphosphorylation in a cell, a tissue or organ that has been removed froman animal. The kit is a packaged combination of one or more containersholding reagents and other items useful for preserving harvested cells,tissues or organs. The kit comprises a container holding one or moreEPACs of the invention. Suitable containers include bottles, bags,vials, test tubes, syringes, and other containers known in the art Forinstance, the kit may comprise a vial containing lyophilized EPAC(s).The kit may also contain other items which are known in the art andwhich may be desirable from a commercial and user standpoint, such as acontainer for the cells, tissue or organ, diluents, buffers, emptysyringes, tubing, gauze pads, disinfectant solution, etc. The kits willalso include instructions for using the kit to contact a cell, tissue ororgan with the EPAC(s) contained in the kit.

D. Oral Care Products and Methods

The EPAC(s) of the invention may also be administered to an animal asoral care products. Oral care products include oral care compositionsand oral care devices. Preferred EPACs for use in the oral care productsof the invention are phosvitins and caseins which are at least partiallydephosphorylated.

Oral care compositions of the invention include washes, rinses, gargles,solutions, drops, emulsions, suspensions, liquids, pastes, gels,ointments, creams, sprays, powders, tablets, gums, lozenges, mints,films, patches, and tooth whitening compositions. Oral care compositionsof the invention include compositions intended for use by consumers andpatients and compositions intended for use by dental professionals(e.g., dental hygienists, dentists and oral surgeons).

The oral care compositions of the invention will comprise an EPAC orEPACs of the invention as active ingredient(s) in admixture with one ormore pharmaceutically-acceptable carriers. The oral care compositions ofthe invention may also comprise one or more other acceptableingredients, including other active compounds and/or other ingredientsconventionally used in oral care compositions. Each carrier andingredient must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to theanimal.

Suitable ingredients, including pharmaceutically-acceptable carriers,for use in oral care compositions, and methods of making and using oralcare compositions, are well known in the art. See, e.g., U.S. Pat. Nos.4,847,283, 5,032,384, 5,043,183, 5,180,578, 5,198,220, 5,242,910,5,286,479, 5,298,237, 5,328,682, 5,407,664, 5,466,437, 5,707,610,5,709,873, 5,738,840, 5,817,295, 5,858,408, 5,876,701, 5,906,811,5,932,193, 5,932,191, 5,951,966, 5,976,507, 6,045,780, 6,197,331,6,228,347, 6,251,372, and 6,350,438, PCT applications WO 95/32707, WO96/08232 and WO 02/13775, and EP applications 471,396, the completedisclosure of all of which are incorporated herein by reference.Conventional ingredients used in oral care compositions include water,alcohols, humectants, surfactants, thickening agents, abrasives,flavoring agents, sweetening agents, antimicrobial agents, anti-cariesagents, anti-plaque agents, anti-calculus agents, pH-adjusting agents,and many others.

The water used in oral care compositions should preferably be of low ioncontent. It should also be free of organic impurities.

The alcohol must be nontoxic. Preferably the alcohol is ethanol. Ethanolis a solvent and also acts as an antibacterial agent and as anastringent.

Humectants suitable for use in oral care compositions include ediblepolyhydric alcohols, such as glycerol, sorbitol, xylitol, butyleneglycol, polyethylene glycol, propylene glycol, mannitol and lactitol.Humectants help keep oral care compositions, such as pastes, fromhardening upon exposure to air, give oral care compositions a moist feelto the mouth, and may impart desirable sweetness.

Surfactants include anionic, nonionic, amphoteric, zwitterionic andcationic synthetic detergents. Anionic surfactants include thewater-soluble salts of alkyl sulfates having 8-20 carbon atoms in thealkyl radical (such as sodium alkyl sulfate), the water-soluble salts ofsulfonated monoglycerides of fatty acids having from 8-20 carbon atoms(such as sodium lauryl sulfate and sodium coconut monoglyceridesulfonates), sarcosinates (such as sodium and potassium salts of lauroylsarcosinate, myristoyl sarcosinate, palmitoyl sarcosinate, stearoylsarcosinate and oleoyl sarcosinate), taurates, higher alkylsulfoacetates (such as sodium lauryl sulfoacetate), isethionates (suchas sodium lauroyl isethionate), sodium laureth carboxylate, sodiumdodecyl benezesulfonate, and mixtures of the foregoing. Preferred arethe sarcosinates since they inhibit acid formation in the mouth due tocarbohydrate breakdown. Nonionic surfactants include poloxamers (soldunder the tradename Pluronic), polyoxyethylene sorbitan esters (soldunder the tradename Tween), fatty alcohol ethoxylates, polyethyleneoxide condensates of alkyl phenols, products derived from thecondensation of ethylene oxide with fatty acids, fatty alcohols, fattyamides, polyhydric alcohols, and polypropyleneoxide, ethylene oxidecondensates of aliphatic alcohols, long-chain tertiary amine oxides,long-chain tertiary phosphine oxides, long-chain dialkyl sulfoxides, andmixtures of such materials. Amphoteric surfactants include betaines(such as cocamidopropylbetaine), derivatives of aliphatic secondary andtertiary amines in which the aliphatic radical can be a straight orbranched chain and wherein one of the aliphatic substituents containsabout 8-18 carbon atoms and one contains an anionic water-solubilizinggroup (such as carboxylate, sulfonate, sulfate, phosphate orphosphonate), and mixtures of such materials. Zwitterionic surfactantsinclude derivatives of aliphatic quaternary ammonium, phosphonium andsulfonium compounds in which the aliphatic radical can be a straight orbranched chain and wherein one of the aliphatic substituents containsabout 8-18 carbon atoms and one contains an anionic water-solubilizinggroup (such as carboxy, sulfonate, sulfate, phosphate or phosphonate).Cationic surfactants include aliphatic quaternary ammonium compoundshaving one long alkyl chain containing about 8-18 carbon atoms (such aslauryl trimethylammonium chloride, cetylpyridinium chloride,cetyltrimethylammonium bromide,diisobuytylphenoxyethyldimethylbenzylammonium chloride, coconutalkyltrimethylammonium nitrite, cetylpyridinium fluoride). Certaincationic surfactants can also act as antimicrobials.

Thickening agents include carboxyvinyl polymers, polyvinylpyrrolidone,polyacrylates, carrageenan, cellulose derivatives (e.g., hydroxypropylcellulose, hydroxypropyl methyl cellulose, methyl cellulose, andhydroxyethyl cellulose), laponite, water-soluble salts of celluloseethers (such as sodium carboxymethylcellulose and sodium carboxymethylhydroxyethyl cellulose), natural gums (such as gum karaya, xanthan gum,gum arabic and gum tragacanth), polymeric polyether compounds (such aspolyethylene oxide and polypropylene oxide), homopolymers of acrylicacid crosslinked with an alkyl ether of pentaerythritol, alkyl ether ofsucrose, carbomers (sold under the tradename Carbopol®), starch,copolymers of lactide and glycolide monomers (the copolymer having anaverage molecular weight of about 1,000-120,000), colloidal magnesiumaluminum silicate and finely divided silica. Thickening agents will beadded in amounts sufficient to give a desired consistency to an oralcare composition.

Abrasives include silicas (including gels and precipitates), aluminas,calcium carbonates, calcium phosphates, dicalcium phosphates, tricalciumphosphates, hydroxyapatites, calcium pyrophosphates, trimetaphosphates,insoluble polymetaphosphates (such as insoluble sodium polymetaphosphateand calcium polymetaphosphate), magnesium carbonates, magnesium oxides,resinous abrasive materials (such as particulate condensation productsof urea and formaldehyde), particulate thermosetting polymerized resins(suitable resins include melamines, phenolics, ureas, melamine-ureas,melamine-formaldehydes, urea-formaldehydes, melamine-urea-formaldehydes,cross-linked epoxides and cross-linked polyesters), and combinations ofthe foregoing. Silica abrasives are preferred because they provideexcellent dental cleaning and polishing performance without undulyabrading tooth enamel or dentine.

Flavoring agents include peppermint, oil, spearmint oil, wintergreenoil, clove, menthol, dihydroanethole, estragole, methyl salicylate,eucalyptol, cassia, 1-menthyl acetate, sage, eugenol, parsley oil,menthone, oxanone, alpha-irisone, alpha-ionone, anise, marjoram, lemon,orange, propenyl guaethol, cinnamon, vanillin, ethyl vanillin, thymol,linalool, limonene, isoamylacetate, benzaldehyde, ethylbutyrate, phenylethyl alcohol, sweet birch, cinnamic aldehyde, cinnamaldehyde glycerolacetal (known as CGA), and mixtures of the foregoing.

Sweetening agents include sucrose, glucose, saccharin, dextrose,levulose, lactose, mannitol, sorbitol, fructose, maltose, xylitol,saccharin salts, thaumatin, aspartame, D-tryptophan, dihydrochalcones,acesulfame, cyclamate salts, and mixtures of the foregoing.

In addition to the flavoring and sweetening agents, the oral carecompositions may include coolants, salivating agents, warming agents andnumbing agents as optional ingredients. Coolants include carboxamides,menthol, paramenthan carboxamides, isopropylbutanamide, ketals, diols,3-1-menthoxypropane-1,2-diol, menthone glycerol acetal, menthyl lactate,and mixtures thereof. Salivating agents include Jambu® (manufactured byTakasago). Warming agents include capsicum and nicotinate esters (suchas benzyl nicotinate). Numbing agents include benzocaine, lidocaine,clove bud oil and ethanol.

Antibacterial and anti-plaque agents include triclosan, sanguinarine andsanguinaria, quaternary ammonium compounds, cetylpyridinium chloride,tetradecylpyridinium chloride and N-tetradecyl-4-ethylpyridiniumchloride, benzalkonium chloride, bisquanides, chlorhexidine,chlorhexidine digluconate, hexetidine, octenidine, alexidine,halogenated bisphenolic compounds,2,2′-methylenebis-(4-chloro-6-bromophenol),5-chloro-2-(2,4-dichlorophenoxy)-phenol, salicylanilide, domiphenbromide, delmopinol, octapinol, other piperadino derivatives, nicin,zinc stannous ion agents, antibiotics (such as augimentin, amoxicillin,tetracycline, doxycycline, minocycline, and metronidazole), analogs andsalts of the foregoing, and mixtures of the foregoing.

Anti-caries agents include sodium fluoride, stannous fluoride, potassiumfluoride, amine fluorides, indium fluoride, sodium monofluorophosphate,calcium lactate, calcium glycerophosphates, strontium salts, andstrontium polyacrylates.

Anti-calculus agents include pyrophosphate salts such as dialkali metalpyrophosphate salts and tetraalkali metal pyrophosphate salts (e.g.,disodium dihydrogen pyrophosphate, tetrasodium pyrophosphate andtetrapotassium pyrophosphate, in their hydrated and unhydrated forms).Other anti-calculus agents which can be used instead of, or in additionto, the pyrophosphate salts include synthetic anionic polymers (such aspolyacrylates and copolymers of maleic anhydride or acid and methylvinyl ether), polyaminopropane sulfonic acid, zinc citrate trihydrate,polyphosphates (such as tripolyphosphate and hexametaphosphate),polyphosphonates (such as disodium ethane-1-hydroxy-1,1-diphosphonate(EHDP), methanedisphosphonic acid, and2-phosphonobutane-1,2,4-tricarboxylic acid), and polypeptides (such aspolyaspartic acid and polyglutamic acid).

The pH of the oral care compositions of the invention should preferablynot be acidic. Thus, the pH of the oral care compositions of theinvention should be greater than about 6.5, preferably from about 7.0 toabout 8.5, more preferably from about 7.2 to about 7.6. Thus, apH-adjusting agent and/or a buffering agent or agents may need to beincluded in the oral care compositions. The pH-adjusting agent may beany compound or mixture of compounds that will achieve the desired pH.Suitable pH-adjusting agents include organic and inorganic acids andbases, such as benzoic acid, citric acid, potassium hydroxide, andsodium hydroxide. Buffering agents include acetate salts, borate salts,carbonate salts, bicarbonate salts (e.g., an alkali metal bicarbonate,such as sodium bicarbonate (also known as baking soda)), gluconates,tartrates, sulfates, citrates (such as sodium citrate), benzoate salts,nitrate salts (such as sodium and potassium nitrate), and combinationsof the foregoing as needed to achieve and maintain the desired pH.

In addition to the one or more EPACs, the oral care compositions of theinvention may include one or more additional anti-inflammatory agents,antioxidants and/or metal-binding compounds.

Suitable anti-inflammatory agents include ibuprofen, flurbiprofen,ketoprofen, aspirin, kertorolac, naproxen, indomethacin, piroxicam,meclofenamic acid, steroids, and mixtures of the foregoing.

Suitable antioxidants include superoxide dismutase, catalase,glutathione peroxidase, ebselen, glutathione, cysteine, N-acetylcysteine, penicillamine, allopurinol, oxypurinol, ascorbic acid,α-tocopherol, Trolox (water-soluble α-tocopherol), vitamin A,β-carotene, fatty-acid binding protein, fenozan, probucol, cyanidanol-3,dimercaptopropanol, indapamide, emoxipine, dimethyl sulfoxide, andothers. See, e.g., Das et al., Methods Enzymol., 233, 601-610 (1994);Stohs, J. Basic Clin. Physiol. Pharmacol., 6, 205-228 (1995).

Suitable metal-binding compounds include metal-binding peptide and/ornon-peptide chelators. Metal-binding peptides and non-peptide chelatorsare known in the art. Preferred are those metal-binding peptides andnon-peptide chelators described in PCT applications WO 01/25265 and WO02/64620, the complete disclosures of which are incorporated herein byreference. Additional metal-binding compounds arepolyethylenepolyamines, such as tetraethylenetriamine (trientine).

The oral care compositions of the invention may advantageously contain aprotease inhibitor to prevent degradation of the EPACs and/or for anadditional therapeutic effect (certain proteases are involved ininflammatory processes and others have been implicated in tissuebreakdown in the mouth). Suitable protease inhibitors includemetalloproteinase and serine protease inhibitors, such as thosedescribed in U.S. Pat. Nos. 6,403,633, 6,350,438, 6,066,673, 5,622,984,and 4,454,338, the complete disclosures of which are incorporated hereinby reference.

Many other ingredients are known that may be incorporated into oral carecompositions. These include suspending agents (such as apolysaccharide—see U.S. Pat. No. 5,466,437), polymeric compounds whichcan enhance the delivery of active ingredients (such as copolymers ofpolyvinylmethylether with maleic anhydride and those delivery enhancingpolymers described in DE 942,643 and U.S. Pat. No. 5,466,437), materialswhich allow for a strong and continuing adherence of the oral carecomposition to the tissues of the mouth, thereby providing for aprotracted topical therapeutic effect (such as natural gums, plantextracts, animal extracts (e.g., gelatin), natural and syntheticpolymers, and starch derivatives; see, e.g., U.S. Pat. Nos. 5,032,384,5,298,237, and 5,466,437), oils, waxes, silicones, coloring agents (suchas FD&C dyes), color change systems, preservatives (such asmethylparaben, propylparaben, and sodium benzoate), opacifying agents(such as titanium dioxide), plant extracts, solubilizing agents (such aspropylene glycol; see, e.g., U.S. Pat. No. 5,466,437), enzymes (such asdextranase and/or mutanase, amyloglucosidase, glucose oxidase withlactoperoxidase, and neuraminidases), synthetic or natural polymers,tooth whitening agents (such from about 0.1% to about 10% by weight of aperoxygen compound; see additional discussion of tooth whiteningcompositions below), an alkali metal bicarbonate (such as sodiumbicarbonate (also known as baking soda), generally present at from about0.01% to about 30% by weight), desensitizers (such as potassium salts(e.g., potassium nitrate, potassium citrate, potassium chloride,potassium tartrate, potassium bicarbonate, and potassium oxalate) andstrontium salts), analgesics (such as lidocaine or benzocaine),anti-fungal agents, antiviral agents, etc.

It will be appreciated that a wide variety of different oral carecompositions can be prepared utilizing the above described ingredientsand other ingredients known in the art or which will be developed. It iswithin the skill in the art to chose appropriate ingredients andcombinations of ingredients and to determine an effective amount of theEPAC(s) of the invention to include in a particular oral carecomposition, given the knowledge in the art and the guidance providedherein.

What follows are a few examples of oral care compositions into which aEPAC or a combination of EPACs could be incorporated. It will beunderstood by those skilled in the art that additional types of oralcare compositions and additional oral care compositions having differentingredients and/or different amounts of ingredients can be preparedutilizing the knowledge and skill in the art and the guidance providedherein.

Dentrifices include toothpastes, tooth gels, tooth powders and liquiddentrifices. Toothpastes and tooth gels generally include a dentalabrasive, a surfactant, a thickening agent, a humectant, a flavoringagent, a sweetening agent, a coloring agent and water. Toothpastes andtooth gels may also include opacifying agents, anti-caries agents,anti-calculus agents, tooth whitening agents, and other optionalingredients. Typically, a toothpaste or tooth gel will contain fromabout 5% to about 70%, preferably from about 10% to about 50%, of anabrasive, from about 0.5% to about 10% of a surfactant, from about 0.1%to about 10% of a thickening agent, from about 10% to about 80% of ahumectant, from about 0.04% to about 2% of a flavoring agent, from about0.1% to about 3% of a sweetening agent, from about 0.01% to about 0.5%of a coloring agent, from about 0.05% to about 0.3% of an anti-cariesagent, from about 0.1% to about 13% of an anti-calculus agent, and fromabout 2% to about 45% water. Tooth powders of course containsubstantially all non-liquid components and typically contain from about70% to about 99% abrasive. Liquid dentrifices may comprise water,ethanol, a humectant, a surfactant, a thickening agent, an abrasive (ifan abrasive is included, a suspending agent (e.g., a high molecularweight polysaccharide) must be included; see U.S. Pat. No. 5,466,437),an antibacterial agent, an anti-caries agent, a flavoring agent and asweetening agent. A typical liquid dentrifice will comprise from about50% to about 85% water, from about 0.5% to about 20% ethanol, from about10% to about 40% of a humectant, from about 0.5% to about 5% of asurfactant, from about 0.1% to about 10% of a thickening agent, and maycontain from about 10% to about 20% of an abrasive, from about 0.3% toabout 2% of a suspending agent, from about 0.05% to about 4% of anantibacterial agent, from about 0.0005% to about 3% of an anti-cariesagent, from about 0.1% to about 5% of a flavoring agent, and from about0.1% to about 5% of a sweetening agent.

Gels include dentrifice gels (see description above), non-abrasive gelsand subgingival gels. Non-abrasive gels and subgingival gels generallyinclude a thickening agent, a humectant, a flavoring agent, a sweeteningagent, a coloring agent, and water. Such gels may also include one ormore anti-caries agents and/or anti-calculus agents. Typically, such agel will contain from about 0.1% to about 20% of a thickening agent,from about 10% to about 55% of a humectant, from about 0.04% to about 2%of a flavoring agent, from about 0.1% to about 3% of a sweetening agent,from about 0.01% to about 0.5% of a coloring agent, and the balancewater. Such gels may also contain from about 0.05% to about 0.3% of ananti-caries agent and from about 0.1% to about 13% of an anti-calculusagent.

Creams generally include a thickening agent, a humectant and asurfactant, and may include a flavoring agent, a sweetening agent, acoloring agent. Typically, a cream will contain from about 0.1% to about30% of a thickening agent, from about 0% to about 80% of a humectant,from about 0.1% to about 5% of a surfactant, from about 0.04% to about2% of a flavoring agent, from about 0.1% to about 3% of a sweeteningagent, from about 0.01% to about 0.5% of a coloring agent, and fromabout 2% to about 45% of water.

Ointments suitable for oral use are described in, e.g., U.S. Pat. Nos.4,847,283, 5,855,872 and 5,858,408, the complete disclosures of whichare incorporated herein by reference. Ointments generally include one ormore of the following: fats, oils, waxes, parafins, silicones,plastibase, alcohols, water, humectants, surfactants, thickening agents,talc, bentonites, zinc oxide, aluminum compounds, preservatives,antiviral compounds, and other ingredients. For instance, the ointmentmay comprise from about 80% to about 90% petrolatum and from about 10%to about 20% ethanol or propylene glycol. As another example, theointment may comprise about 10% petrolatum, about 9% lanolin, about 8%talc, about 32% cod liver oil, and about 40% zinc oxide. As a thirdexample, the ointment may comprise from about 30% to about 45% water,from about 10% to about 30% oil (e.g., petrolatum or mineral oil), fromabout 0.1% to about 10% emulsifier (e.g., wax NF), from about 2% toabout 20% humectant (e.g., propylene glycol), from about 0.05% to about2% preservatives (e.g., methyl paraben and propyl paraben), and fromabout 10% to about 40% sterol alcohol.

Mouthwashes, rinses, gargles and sprays generally include water,ethanol, and/or a humectant, and preferably also include a surfactant, aflavoring agent, a sweetening agent, and a coloring agent, and mayinclude a thickening agent and one or more anti-caries agents and/oranti-calculus agents. A typical composition contains from about 0% toabout 80% of a humectant, from about 0.01% to about 7% of a surfactant,from about 0.03% to about 2% of a flavoring agent, from about 0.005% toabout 3% of a sweetening agent, from about 0.001% to about 0.5% of acoloring agent, with the balance being water. Another typicalcomposition contains from about 5% to about 60%, preferably from about5% to about 20%, ethanol, from about 0% to about 30%, preferably fromabout 5% to about 20%, of a humectant, from about 0% to about 2%emulsifying agents, from about 0% to about 0.5% of a sweetening agent,from about 0% to about 0.3% of a flavoring agent, and the balance water.A further typical composition contains from about 45% to about 95%water, from about 0% to about 25% ethanol, from about 0% to about 50% ofa humectant, from about 0.1% to about 7% of a surfactant, from about0.1% to about 3% of a sweetening agent, from about 0.4% to about 2% of aflavoring agent, and from about 0.001% to about 0.5% of a coloringagent. These compositions may also comprise from about 0.05% to about0.3% of an anti-caries agent, and from about 0.1% to about 3% of ananti-calculus agent

Solutions generally include water, a preservative, a flavoring agent,and a sweetening agent, and may include a thickening agent and/or asurfactant. Typically, solutions contain from about 85% to about 99%water, from about 0.01% to about 0.5% of a preservative, from about 0%to about 5% of a thickening agent, from about 0.04% to about 2% of aflavoring agent, from about 0.1% to about 3% of a sweetening agent, andfrom about 0% to about 5% of a surfactant.

Lozenges and mints generally include a base, a flavoring agent and asweetening agent. The base may be a candy base (hard sugar candy),glycerinated gelatin or a combination of sugar with sufficient mucilageto give it form. See U.S. Pat. No. 6,350,438 and Remington, The ScienceAnd Practice Of Pharmacy, 19^(th) edition (1995). Lozenge compositionsalso typically include one or more fillers (e.g., a compressible sugar)and lubricants.

Chewing gums, chewable tablets and chewable lozenges are described inU.S. Pat. Nos. 6,471,991, 6,296,868, 6,146,661, 6,060,078, 5,869,095,5,709,873, 5,476,647, and 5,312,626, PCT applications WO 84/04453 and WO99/02137, and Lieberman et al., Pharmaceutical Dosage Forms, 2^(nd) ed.(1990), the complete disclosures of which are incorporated here in byreference.

As one example, a compressed chewable tablet comprises awater-disintegratable, compressible carbohydrate (such as mannitol,sorbitol, maltitol, dextrose, sucrose, xylitol, lactose and mixturesthereof), a binder (such as cellulose, cellulosic derivatives, polyvinylpyrrolidone, starch, modified starch and mixtures thereof), and,optionally, a lubricant (such as magnesium stearate, stearic acid, talc,and waxes), sweetening, coloring and flavoring agents, a surfactant, apreservative, and other ingredients. All of the ingredients, includingthe EPAC(s) of the invention, are dry blended and compressed into atablet.

As another example, a chewable tablet may comprise a core surrounded byan outer layer wrapping the core. The core may comprise a EPAC or EPACsof the invention and, optionally, other active ingredients in a jellybase or a chewable base. The outer layer may be a chewable base. Thejelly base may comprise pectin, sorbitol, maltitol, isomalt, liquidglucose, sugar, citric acid and/or a flavoring agent. The chewable baseof the core or outer layer may be a gum, soft candy, nougat, caramel orhard candy. The tablets are formed by extrusion of the core and outerlayer to form a rope, followed by cutting the rope into tablets.

Chewing gum compositions generally include a gum base, a flavoring agentand a sweetening agent. Suitable gum bases include jelutong, rubber,latex, chicle, and vinylite resins, desirably with conventionalplasticizers or softeners. Plasticizers include triacetin, acetyltributyl citrate, diethyl sebacetate, triethyl citrate, dibutylsebacetate, dibutyl succinate, diethyl phthalate and acetylatedmonoglycerides. Typically, chewing gum compositions contain from about50% to about 99% gum base, from about 0.4% to about 2% of a flavoringagent and from about 0.01% to about 20% of a sweetening agent. TheEPAC(s) of the invention and other active ingredients may beincorporated into a gum base by, e.g., stirring them into a warm gumbase or coating them onto the outer surface of the gum base.

Films and sheets, and gels which form solids in the mouth, made oflactide/glycolide copolymers are described in U.S. Pat. Nos. 5,198,220,5,242,910 and 6.350,438. Another polymer film suitable for use in themouth is described in PCT application WO 95/32707. Patches that adhereto hard dental surfaces, such as teeth and dentures, and which degradein the mouth, are described in U.S. Pat. No. 6,197,331. All of thesematerials slowly release active agents contained in them into the mouth.Other compositions (including pastes, gels, ointments, liquids andfilms) providing for slow release of active agents are also known. See,e.g., U.S. Pat. Nos. 5,032,384, 5,298,237, 5,466,437, 5,709,873, and6,270,781.

Tooth whitening compositions will comprise a tooth whitening agent.Tooth whitening agents include peroxides, percarbonates and perboratesof the alkali and alkaline earth metals or complex compounds containinghydrogen peroxide. Tooth whitening agents also include peroxide salts ofthe alkali or alkaline earth metals. The most commonly used toothwhitening agent is carbamide peroxide. Other commonly used toothwhitening agents are hydrogen peroxide, peroxyacetic acid and sodiumperborate. These tooth whitening agents liberate active oxygen andhydrogen peroxide. Tooth whitening agents can be present in toothwhitening compositions at a concentration of from about 0.1% to about90%; typically, the concentration of carbamide peroxide in toothwhitening compositions is from about 10% to about 25%.

Many tooth whitening compositions are known in the art, includingaqueous solutions, gels, pastes, liquids, films, strips, one-partsystems, two-part systems, compositions that require activation of thetooth whitening agent (e.g., by inclusion of a radiant-energy orheat-energy absorbing substance, such as substantially conjugatedhydrocarbons, which activates the bleaching agent when irradiated), etc.See, e.g., U.S. Pat. Nos. 5,302,375, 5,785,887, 5,858,332, 5,891,453,5,922,307, 6,322,773, 6,419,906, and PCT applications WO 99/37236, WO01/89463 and WO 02/07695, the complete disclosures of which areincorporated herein by reference. Also, many other oral carecompositions (e.g., toothpastes) and devices (e.g., dental flosses)comprise a tooth whitening agent.

The use of tooth whitening compositions, or of one of the many oral carecompositions and devices which comprise a tooth whitening agent, resultsin the production of reactive oxygen species (ROS) and can causeinflammation of the tissues of the mouth. Incorporation of an EPAC orEPACs in tooth whitening compositions or other oral care compositionsand devices comprising a tooth whitening agent will reduce or preventthe inflammation and should also reduce the production of ROS (as notedabove, once phosphorylated, the EPACs bind metal ions and, as a result,can reduce the production of ROS). The inclusion of a EPAC or EPACs insuch compositions may also result in more effective whitening, sincehydrogen peroxide (which is responsible for the whitening of teeth bythe hydrogen peroxide-type whitening agents and which is converted intohydroxyl radicals in the presence of metal ions) may not be convertedinto hydroxyl radicals (because of the binding of metal ions byphosphorylated EPACs) and will, therefore, remain active longer.Alternatively, an oral care composition or device comprising an EPAC orEPACs can be used before or after the tooth whitening composition ororal care composition or device comprising a tooth whitening agent toreduce or prevent the inflammation and, possibly, the production of ROS.

For instance, teeth are commonly whitened by applying a tooth whiteningcomposition to the teeth by means of a dental tray or trough. An EPAC orEPACs of the invention could be incorporated into the tooth whiteningcomposition that is used in the tray or trough. Alternatively, aseparate composition comprising an EPAC or EPACs of the invention couldbe applied to the teeth in a cleaned or different tray or trough afterthe application of the tooth whitening composition is completed. In afurther alternative, a wash or rinse comprising an EPAC or EPACs of theinvention could be used to rinse the mouth before and/or after theapplication of the tooth whitening composition.

A recently developed product for applying a tooth whitening compositionto the teeth is a flexible strip. See, e.g., U.S. Pat. Nos. 5,891,453and 6,419,906. An EPAC or EPACs could be incorporated into such strips.For instance, the EPAC(s) could be incorporated into the tooth whiteningcomposition, which is then applied to the strips, or a solution, gel orother composition comprising the EPAC(s) could be separately applied tothe strips, either during their manufacture or just prior to use by thepatient. In yet another alternative, strips comprising a tooth whiteningcomposition and strips comprising the EPAC(s) could both be supplied tothe patient and would be used sequentially.

The oral care compositions of the invention may comprise a single phaseor a plurality of phases. A plurality of phases will be used, e.g.,where some of the ingredients are incompatible, some of the ingredientsare unstable, or the ingredients are best combined at the time of use.Thus, one of the phases will include some of the ingredients, and theremainder of the ingredients will be contained in one or more additionalphases. The plurality of phases may be a plurality of separatecompositions, in which case the plurality of phases will be provided ina plurality of separate containers or in a plurality of compartments ina single container, and the plurality of phases will be combined at thetime of use. As an alternative, the plurality of phases may be formed byencapsulating some of the ingredients, in which case the plurality ofphases may all be contained in a single container. Multi-phase oral carecompositions are described in, e.g., U.S. Pat. Nos. 5,302,375,5,906,811, 5,976,507, 6,228,347 and 6,350,438 and PCT application numberWO 99/37236.

The invention also provides oral care devices comprising an EPAC orEPACs. Oral care devices of the invention include devices intended foruse by consumers and patients and devices intended for use by dentalprofessionals (e.g., dental hygienists, dentists and oral surgeons).

The oral care devices of the invention include surgical materials (suchas sutures and sponges), flosses, tapes, chips, strips, fibers, atoothpick or rubber tip, dental implants and dental appliances (such astrays and troughs that fit over and cover the teeth and, optionally, theperiodontal tissue) having an EPAC or EPACs adhered to, absorbed into,bound to, attached to, entrapped in, coated onto, or otherwiseincorporated into, them. See, e.g., U.S. Pat. Nos. 5,709,873, 5,863,202,5,891,453, 5,967,155, 5,972,366, 5,980,249, 6,026,829, 6,080,481,6,102,050, 6,350,438, 6,419,906, PCT application WO 02/13775, and EPapplication 752833, which describe such oral care devices and methods ofincorporating compounds into them (the complete disclosures of all ofthese patents and applications are incorporated herein by reference).For instance, an EPAC or EPACs can be incorporated into a binder (e.g.,a wax or polymer) and coated onto dental floss, dental floss can besoaked in a bath of a liquid containing an EPAC or EPACs to impregnateor coat the floss with the compound(s), an EPAC or EPACs in solid (e.g.,freeze-dried) form can be incorporated into a polymer film suitable forapplication to the teeth, an EPAC or EPACs in a solution or gel can beapplied to a flexible strip suitable for application to teeth, or asuture or other surgical material can be soaked in a solution containingan EPAC or EPACs followed by removal of the solvent so that thecompound(s) become associated with (bound to, entrapped in, coated onto,etc.) the suture or surgical material. See, e.g., U.S. Pat. Nos.5,891,453, 5,967,155, 5,972,366, 6,026,829, 6,080,481, 6,102,050, and6,419,906.

Also included within the scope of the invention are oral care productsfor animals, such as foods, chews, and toys. Suitable products aredescribed in U.S. Pat. No. 6,350,438.

An EPAC or EPACs of the invention can be used to treat a tissue of ananimal's mouth to inhibit increased phosphorylation. “Mouth” is usedherein to mean the cavity bounded externally by the lips and internallyby the pharynx that encloses the tongue, gums and teeth. Thus, thetissues of the mouth include the lips, tongue, gums, buccal tissue,palate and teeth. A single tissue, a plurality of tissues, a portion ofone or more tissues, all or substantially all of the tissues of themouth, or combinations of the foregoing, may be treated according to theinvention.

To treat a tissue of the mouth, the tissue is contacted with an EPAC orEPACs of the invention. For instance, the tissue may be contacted withan oral care composition comprising the EPAC(s). Methods of contactingtissues of the mouth with oral care compositions are well known in theart. Suitable methods include rinsing the tissue with a solution (e.g.,a mouthwash, rinse, spray, liquid dentrifice, or other solution),brushing the teeth with a dentrifice (e.g., a toothpaste, tooth gel, orpowder), applying a non-abrasive solution, gel, paste, cream or ointmentdirectly to the tissue (with or without the use of an applicator),chewing gum, chewing or sucking a lozenge, mint or tablet, and manyother means of topical application. Suitable applicators for applyingoral care compositions, such as solutions, gels, pastes, creams andointments, to a tissue include a swab, a stick, a plastic paddle, adropper, a syringe, a strip (such as those described in U.S. Pat. Nos.5,891,453 and 6,419,906), a finger, or a dental tray or appliance (suchas those shown in U.S. Pat. Nos. 5,863,202 and 5,980,249 and EPapplication 752833) which allows for immersion of the teeth and,optionally, the periodontal tissue in, e.g., a gel or solution. Inaddition, to treat a tissue of the mouth, the tissue may be contactedwith an oral care device comprising the EPAC(s). Methods of contactingtissues of the mouth with oral care devices are well known in the art.For instance, sutures can be used to close a surgical wound or a woundresulting from a tooth extraction, dental floss can be used to floss theteeth, etc.

The treatment of the tissue can be prophylactic treatment. For instance,the tissue may be treated as part of a prophylactic oral care regimen.The EPAC(s) can be incorporated into an oral care composition or device,such as a toothpaste, a tooth gel, a mouthwash or rinse, or a dentalfloss, that is employed in such a regimen and will be used regularly,preferably at least once per day, more preferably two or three times perday. In another alternative, the EPAC(s) may be contained in a separateoral care composition or device which will be used separately from othercompositions and devices employed in the prophylactic oral care regimen.For instance, the EPAC(s) can be incorporated into a mouthwash or rinse,a gum, a lozenge or a chewable tablet, which would be used regularly,preferably at least once per day, more preferably at least two or threetimes per day.

Tissues may also be treated prophylactically in connection with avariety of dental procedures, including surgeries and tooth extractions.For instance, the tissue(s) on which surgery is being performed, thosetissues near the area where the surgery is being performed or, for easeof treatment, all or substantially of the tissues of the mouth, can betreated prior to surgery, during surgery, after the surgery, orcombinations thereof. Similarly for a tooth extraction, the tissue(s)surrounding the tooth which is to be extracted, adjacent tissues or, forease of treatment, all or substantially of the tissues of the mouth, canbe treated prior to tooth extraction, during the tooth extraction, afterthe tooth extraction, or combinations thereof. For instance, the mouthcould be rinsed prior to surgery or tooth extraction with a solutioncomprising the EPAC(s), the wound(s) caused by the surgery or toothextraction could be closed with sutures having the EPAC(s) incorporatedinto them, and/or the mouth could be rinsed immediately after thesurgery or tooth extraction, and/or at intervals thereafter, with asolution comprising the EPAC(s). Finally, as described above, tissuesmay be treated prophylactically in connection with the whitening of theteeth of an animal.

An EPAC or EPACs of the invention can also be used to treat a disease orcondition of a tissue of an animal's mouth that is mediated by increasedphosphorylation. Specific diseases and conditions treatable according tothe invention include inflammation and inflammatory diseases andconditions, such as inflammation of the periodontal tissue, gingivitis,periodontitis, infections (bacterial infections, viral infections, yeastinfections and fungal infections), ulcers, cold sores, canker sores andinflammation accompanying surgery or tooth extraction. The treatment ofother diseases and conditions of the mouth, such as cancer, is moretypically performed by, or under the supervision of, a medical doctor,rather than a dentist. Accordingly, the treatment of these disease andconditions was dealt with above in the discussion of therapeutic methodsand pharmaceutical products. However, the use of the oral care productsof the invention and the use of the pharmaceutical products of theinvention together in the treatment of these types of diseases andconditions of the mouth should be beneficial.

It is understood by those skilled in the art that the dosage amount ofthe EPAC(s) needed to treat a tissue of an animal's mouth will vary withthe particular EPAC employed, whether the treatment is prophylactic orfor the treatment of a disease or condition, the identity of the diseaseor condition to be treated, the severity of the disease or condition,the type of oral care composition used, the duration of the treatment,the identify of any other drugs being administered to the animal, theage, size and species of the animal, and like factors known in themedical and veterinary arts. In general, a suitable daily dose of acompound of the present invention will be that amount of the compoundwhich is the lowest dose effective to produce a therapeutic effect. Itis expected that usage of oral care compositions comprising from about0.000001% to about 20% of an EPAC or EPACs one or more times per daywill provide effective daily dosages. However, the actual daily dosageto be employed, the number of treatments per day, and the length oftreatment will be determined by an attending dentist or veterinarianwithin the scope of sound medical judgment.

The invention also provides a kit comprising an oral care productaccording to the invention. In the case where the oral care product isan oral care composition, the kit may also include an applicator forapplying the oral care composition to a tissue of an animal's mouth,such as a swab, a stick, a plastic paddle, a dropper, a syringe, a strip(such as that described in U.S. Pat. Nos. 5,891,453 and 6,419,906) or adental tray or appliance (such as those shown in U.S. Pat. Nos.5,863,202 and 5,980,249 and EP application 752833) which allows forimmersion of the teeth and, optionally, the periodontal tissue in, e.g.,a gel or solution. The kit could also include a cup, vial or otherdevice for dispensing and/or measuring the amount of the oral carecomposition of the invention needed for the intended use. Of course, thekits could include both an oral care composition and an oral care deviceaccording to the invention. In addition to an oral care compositionand/or device of the invention, the kits could also comprise anothertype of oral care composition or device, such as a tooth whiteningcomposition, strips comprising a tooth whitening agent, applicators forapplying oral care compositions, etc. Kits according to the inventionwill also include instructions for using the kit and/or the oral careproduct of the invention and may include any other desired items.

E. Personal Care Products and Methods

The EPAC(s) of the invention may also be administered to an animal aspersonal care products. Personal care products include personal carecompositions and personal care devices.

Personal care compositions and devices of the invention includecompositions and devices intended for use by consumers and patients andcompositions and devices intended for use by professionals (e.g.,dermatologists, beauty salons and spas). Preferred EPACs for use in thepersonal care products of the invention are phosvitins and caseins whichare at least partially dephosphorylated.

Personal care compositions include cosmetics, skin creams and lotions,face and body moisturizers, suntan creams and lotions, oils, washes,rinses, solutions, eye drops, emulsions, liquids, gels, ointments,sprays, powders, deodorants, shampoos, scalp treatment compositions, lipglosses, lip balms, anti-acne preparations, analgesics, etc.

The personal care compositions of the invention comprise an EPAC orEPACs and a pharmaceutically-acceptable carrier. The personal carecompositions may also comprise one or more other acceptable ingredients,including other active compounds and/or other ingredients conventionallyused in personal care compositions. Each carrier and ingredient must be“acceptable” in the sense of being compatible with the EPAC(s) and anyother ingredients of the composition and not being injurious to theanimal. Suitable ingredients for use in personal care compositions andmethods of making and using personal care compositions are well known inthe art.

A wide variety of carriers suitable for use in skin care compositionsare well known in the art. For example, emulsion carriers (includingoil-in-water, water-in-oil, water-in-oil-in-water andoil-in-water-in-silicone emulsions) can be used. These emulsions cancover a broad range of viscosities (e.g., from about 100 centipoise(cps) to about 200,000 cps). Other suitable carriers include: anhydrousliquid solvents, such as oils, alcohols and silicones (e.g., mineraloil, ethanol, isopropanol, dimethicone, cyclomethicone and the like);aqueous-based single phase liquid solvents (e.g., hydro-alcoholicsolvent systems); and thickened versions of these anhydrous andaqueous-based single phase solvents (e.g., where the viscosity of thesolvent has been increased to form a solid or semi-solid by the additionof appropriate gums, resins, waxes, polymers, salts and the like). Thecarrier preferably comprises from about 50% to about 99% by weight ofthe skin care compositions, more preferably from about 75% to about 99%,most preferably from about 85% to about 95%.

A wide variety of carriers suitable for use in hair care compositionsare also well known in the art. For instance, water, alcohols (e.g.,methanol, ethanol and isopropanol) and mixtures thereof can be used. Thecarriers can also comprise a wide variety of additional materialsincluding acetone, hydrocarbons (e.g., isobutane, hexane, decene),linalool, esters (e.g., ethyl acetate and dibutyl phthalate), volatilesilicone derivatives (e.g., siloxanes, such as phenyl pentamethyldisiloxane, methoxypropyl heptamethyl cyclotetrasiloxane, chloropropylpentamethyl disiloxane, hydroypropyl pentamethyl disiloxane, octamethylcyclotetrasiloxane, decamethyl cyclopentasiloxane, cyclomethicone anddimethicone), and mixtures thereof. Hair care products having a lowviscosity may also utilize an emulsifying agent (preferably at a levelof from about 0.01% to about 7.5% by weight of the composition). Thecarrier will comprise from about 0.5% to about 99.5% by weight of thehair care compositions, preferably from about 5.0% to about 99.5%, morepreferably from about 10.0% to about 98.0%.

In addition to the EPAC(s) and the carrier, the personal carecompositions of the invention can comprise a wide variety of additionalingredients. These additional ingredients include pharmaceuticallyactive ingredients (e.g., anti-acne actives, analgesic actives,antipruritic actives, anesthetic actives and antimicrobial actives),other active ingredients (e.g., sunscreening actives, sunless tanningactives, skin bleaching actives, anti-dandruff actives, antiperspirantactives and deodorant actives), conditioners, humectants, moisturizers,surfactants, thickeners, emollients and other ingredients commonly usedin personal care compositions.

As noted above, the pharmaceutically active ingredients that can beincluded in the personal care compositions of the invention in additionto the EPACs include anti-acne actives, analgesic actives, antipruriticactives, anesthetic actives and antimicrobial actives. Amounts of theseingredients to include in the compositions are known in the art or canbe determined empirically. Suitable dosage amounts will vary with, e.g.,the specific active ingredient, the ability of the compositions topenetrate the active through the skin, the amount of composition to beapplied, the particular condition being treated, the age and physicalcondition of the animal being treated, the severity of the condition,the duration of the treatment, the nature of concurrent therapy and likefactors.

Anti-acne actives include the keratolytics (such as salicylic acid,sulfur, lactic acid, glycolic, pyruvic acid, urea, resorcinol andN-acetylcysteine), retinoids (such as retinoic acid and itsderviatives), antibiotics and antimicrobials (such as benzoyl peroxide,octopirox, erythromycin, zinc, tetracycline, triclosan, azelaic acid andits derivaties, phenoxy ethanol, phenoxy propanol, ethylacetate,clindamycin and meclocycline), sebostats (such as flavinoids), alpha andbeta hydroxy acids, and bile salts (such as scymnol sulfate and itsderivatives, deoxycholate and cholate).

Analgesic actives include salicylic acid derivatives (such as methylsalicylate), species and derivatives of the genus capsicum (such ascapsaicin), steroids (such as hydrocortisone) and non-steroidalanti-inflammatory drugs (NSAIDS). The NSAIDS can be selected from thefollowing categories: propionic acid derivatives (aspirin,acetaminophen, ibuprofen, naproxen, benoxaprofen, flurbiprofen,fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen,oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen,tiaprofenic acid, fluprofen and bucloxic acid), acetic acid derivatives,fenamic acid derivatives, biphenylcarboxylic acid derivatives andoxicams.

Antipruritic actives include the pharmaceutically-acceptable salts ofmethdilizine and trimeprazine.

Anesthetic actives include the pharmaceutically-acceptable salts oflidocaine, bupivacaine, chlorprocaine, dibucaine, etidocaine,mepivacaine, tetracaine, dyclonine, hexylcaine, procaine, cocaine,ketamine, pramoxine and phenol.

Antimicrobial (antibacterial, antifungal, antiprotozoal and antiviral)actives include pharmaceutically-acceptable salts of β-lactams,quinolones, ciprofloxacin, norfloxacin, tetracycline, erythromycin,amikacin, triclosan, doxycycline, capreomycin, chlorhexidine,chlortetracycline, oxytetracycline, clindamycin, ethambutol,metronidazole, pentamidine, gentamicin, kanamycin, lineomycin,methacycline, methenamine, minocycline, neomycin, netilmicin,paromomycin, streptomycin, tobramycin, miconazole, amanfadine,octopirox, parachlorometa xylenol, nystatin, tolnaftate andclotrimazole.

Sunscreening agents include 2-ethylhexyl p-methoxycinnamate,2-ethylhexyl N,N-dimethyl-p-aminobenzoate, p-aminobenzoic acid,2-phenylbenzimidazole-5-sulfonic acid, octocrylene, oxybenzone,homomethyl salicylate, octyl salicylate,4,4′-methoxy-t-butyldibenzoylmethane, 4-isopropyl dibenzoylmethane,3-benzylidene camphor, 3-(4-methylbenzylidene) camphor, titaniumdioxide, zine oxide, silica, iron oxide, and mixtures thereof.Additional sunscreening agents include those having, in a singlemolecule, two distinct chromophore moities which exhibit differentultraviolet radiation absorption spectra (one absorbs predominantly inthe UVA range and one absorbs predominantly in the UVB range), such as4-N,N-(2-ethylhexyl)methylaminobenzoic acid ester of2,4-dihydroxybenzophenone, 4-N,N-(2-ethylhexyl)methylaminobenzoic acidester of 4-hydroxydibenzoylmethane,4-N,N-(2-ethylhexyl)methylaminobenzoic acid ester of2-hydroxy-4-(2-hydroxyethoxy)benzophenone,4-N,N-(2-ethylhexyl)methylaminobenzoic acid ester of4-(2-hydroxyethoxy)dibenzoylmethane, and mixtures thereof. See also PCTapplication WO 03/013468 which describes additional suitablesunscreening agents. Generally, the sunscreens will comprise from about0.5% to about 20% by weight of the compositions. Exact amounts will varydepending upon the sunscreen chosen and the desired Sun ProtectionFactor (SPF), SPF is a commonly used measure of photoprotection of asunscreen against erythema. See Federal Register, Volume 43, No. 166,pages 38206-38269, Aug. 25, 1978.

Sunless tanning actives include dihydroxyacetone, glyceraldehyde,indoles and their derivatives, and the like.

Skin bleaching actives include hydroquinone, ascorbic acid, kojic acidand sodium metabisulfite.

Anti-dandruff actives include zinc pyrithione, octopirox, seleniumdisulfide, sulfur, coal tar and the like.

Antiperspirant actives include astringent metallic salts, such as theinorganic and organic salts of aluminum, zirconium and zinc, as well asmixtures thereof.

Deodorant actives include bacteriostats (e.g.,2,2′-methylenebis(3,4,6-trichlorophenol),2,4,4′-trichloro-T-hydroxy(diphenyl ether) (also known as triclosan),zinc phenolsulfonate, 2,2′-thiobis(4,6-dichlorophenol),p-chloro-m-xylenol, dichloro-m-xylenol, sodium N-lauroyl sarcosine,sodium N-palmitoyl sarcosine, lauroyl sarcosine, N-myristoyl glycine,potassium N-lauroyl sarcosine, aluminum chlorhydroxy lactate, and thelike).

Conditioning agents useful in the compositions, especially the hair carecompositions, include hydrocarbons, silicone fluids and cationicmaterials. The hydrocarbons can be either straight or branched-chain andcan contain from about 10 to about 16 carbon atoms. Examples of suitablehydrocarbons include decane, dodecane, tetradecane, tridecane andmixtures thereof. Silicone conditioning agents include cyclic or linearpolydimethylsiloxanes, phenyl and alkyl phenyl silicones, and siliconecopolyols. Cationic conditioning agents include quaternary ammoniumsalts (e.g., dialkyl dimethyl ammonium salts wherein the alkyl groupshave 12-22 carbon atoms (such as ditallow dimethyl ammonium chloride,ditallow dimethyl ammonium methyl sulfate, dihexadecyl dimethyl ammoniumchloride and di(hydrogenated tallow) ammonium chloride) and dicationics(such as tallow propane diammonium dichloride)), quaternaryimidazolinium salts (e.g., imidazolinium salts containing alkyl groupscontaining 12-22 carbon atoms (such as1-methyl-1[(stearoylamide)ethyl]-2-heptadecyl-4,5-dihydroimidazoliniumchloride,1-methyl-1[(palmitoylamide)ethyl]-2-octadecyl-4,5-dihydroimidazoliniumchloride and 1-methyl-1[(tallowamide)ethyl]-2-tallow-imidazoliniummethyl sulfate)) and the salts of fatty amines (e.g., stearylaminehydrochloride, soyamine hydrochloride and stearylamine formate).

Humectants and moisturizing agents include urea, guanidine, glycolicacid and glycolate salts (e.g., ammonium and quaternary alkyl ammonium),lactic acid and lactate salts (e.g, ammonium and quaternary alkylammonium), aloe vera in any of its variety of forms (e.g., aloe veragel), polyhydroxy alcohols (e.g, sorbitol, glycerol, hexanetriol,propylene glycol, butylene glycol, hexylene glycol and the like)),polyethylene glycols, sugars and starches, sugar and starch derivatives(e.g., alkoxylated glucose), hyaluronic acid, lactamidemonoethanolamine, acetamide monoethanolamine, and mixtures thereof.These agents will generally be present at a level of from about 0.1% toabout 20% of the weight of the compositions.

Surfactants useful in the compositions include anionic, nonionic,cationic, zwitterionic and amphoteric surfactants. Suitable anionicsurfactants include long chain sulfates, sulfonates, isethionates,carboxylates, taurates, and sulfosuccinates, such as alkyl glycerylether sulfonate, ammonium lauryl sulfate, ammonium laureth sulfate,triethylamine lauryl sulfate, triethylamine laureth sulfate,triethanolamine lauryl sulfate, triethanolamine laureth sulfate,monoethanolamine lauryl sulfate, monoethanolamine laureth sulfate,diethanolamine lauryl sulfate, diethanolamine laureth sulfate, lauricmonoglyceride sodium sulfate, sodium lauryl sulfate, sodium laurethsulfate, potassium lauryl sulfate, potassium laureth sulfate, sodiumlauryl sarcosinate, sodium lauroyl sarcosinsate, lauryl sarcosine,cocoyl sarcosine, ammonium cocoyl sulfate, ammonium lauroyl sulfate,sodium cocoyl sulfate, sodium lauroyl suflate, potassium cocoyl sulfate,potassium lauryl sulfate, triethanolamine lauryl sulfate,monoethanolamine cocoyl sulfate, monoethanolamine lauryl sulfate, sodiumtridecyl benzene sulfonate and sodium dodecyl benzene sulfonate. Forcationic surfactants, see U.S. Pat. No. 5,916,548 and the referencescited therein. Nonionic surfactants include the compounds produced bycondensation of alkylene oxide groups (hydrophilic in nature) with anorganic hydrophobic compound, which may be aliphatic or alkyl aromaticin nature. Amphoteric and zwitterionic surfactants include betaines,such as amidocarboxybetaines, alkyl betaines, amidopropyl betaines,amidopropyl sultaines and sulfobetaines. Additional amphoteric andzwitterionic surfactants include derivatives of aliphatic quaternaryammonium and sulfonium compounds, in which the aliphatic radicals can bestraight or branched chain and wherein one of the aliphatic substituentscontains from about 8-18 carbon atoms and one contains an anionicwater-solubilizing group (e.g., carboxy, sulfonate or sulfate). Furtheramphoteric and zwitterionic surfactants include derivatives of aliphaticsecondary and tertiary amines, in which the aliphatic radicals can bestraight or branched chain and wherein one of the aliphatic substituentscontains from about 8-18 carbon atoms and one contains an anionicwater-solubilizing group (e.g., carboxy, sulfonate or sulfate), such assodium 3-dodecyl-aminopropionate, sodium 3-dodecylamino propanesulfonate and N-alkyl taurines. The surfactant or mixture of surfactantswill generally be present at a level of from about 0.2% to about 30% ofthe weight of the compositions.

Thickeners include carboxylic acid polymers (described in U.S. Pat. No.5,916,548, the complete disclosure of which is incorporated herein byreference). These crosslinked polymers contain one or more monomersderived from acrylic acid, substituted acrylic acids and salts andesters of these acrylic acids and the substituted acrylic acids, whereinthe crosslinking agent contains two or more carbon-carbon double bondsand is derived from a polyhydric alcohol. Specific examples of suchpolymers are the carbomers, which are homopolymers of acrylic acidcrosslinked with allyl ethers of sucrose or pentaerytritol (available asthe Carbopol® 900 series from B.F. Goodrich), and copolymers of C₁₀₋₃₀alkyl acrylates with one or more monomers of acrylic acid, methacrylicacid or one of their short chain (C₁₋₄ alcohol) esters, wherein thecrosslinking agent is an allyl ether of sucrose or pentaerytritol (alsoknown as acrylates/C10-30 alkyl acrylate crosspolymers and available asCarbopol® 1342, Pemulen TR-1 and Pemulen TR-2 from B.F. Goodrich). Otherthickeners include xanthan gum, guar gum, carboxymethyl cellulose,hydroxymethyl cellulose, hydroxyethyl cellulose, alkyl modifiedhydroxyalkyl celluloses (e.g, long chain alkyl modified hydroxyethylcelluloses, such as cetyl hydroxyethyl cellulose) and magnesium aluminumsilicate. These thickeners will generally be present at a level of fromabout 0.025% to about 1% of the weight of the compositions.

Emulsifiers suitable for use in personal care compositions can be any ofa wide variety of nonionic, cationic, anionic and zwitterionicemulsifiers. Examples of suitable emulsifiers include esters ofglycerin, esters of propylene glycol, fatty acid esters of polyethyleneglycol, fatty acid esters of polypropylene glycol, esters of sorbitol,esters of sorbitan anhydrides, carboxylic acid copolymers, esters andethers of glucose, ethoxylated ethers, ethoxylated alcohols, fatty acidamides, acyl lactylates, soaps, and mixtures thereof. Specific suitableemulsifiers include polyethylene glycol 20 sorbitan monolaurate(Polysorbate 20), polyethylene glycol 5 soya sterol, Steareth-20,Ceteareth-20, PPG-2 methyl glucose ether distearate, Ceteth-10,Polysorbate 80, Polysorbate 60, glyceryl stearate, PEG-100 stearate andmixtures thereof. The emulsifiers will generally be present at a levelof from about 0.1% to about 10% of the weight of the compositions.

Emollients include volatile and nonvolatile silicone oils, highlybranched hydrocarbons and nonpolar carboxylic acid and alcohol esters,and mixtures thereof. The emollients will generally be present at alevel of from about 1% to about 50% of the weight of the compositions.

A variety of additional ingredients can be included in the personal carecompositions. These additional ingredients include vitamins andderivatives thereof (e.g., ascorbic acid, vitamin E tocopheryl acetate,retinoic acid, retinol, retinoids and the like), polyquatemium andmineral oil, resins, gums, polymers for aiding in the film-formingproperties and substantivity of the composition (such as a copolymer ofeicosene and vinyl pyrrolidone), suspending agents (e.g., ethyleneglycol distearate and the like), preservatives, skin penetration aids,antioxidants, chelators, sequestrants and aesthetic components (e.g.,fragrances, colorings, essential oils, skin sensates, astringents andskin soothing agents; specific examples of such aesthetic componentsinclude panthenol and its derivatives, pantothenic acid and itsderivatives, clove oil, menthol, camphor, eucalyptus oil, eugenol,menthyl lactate, witch hazel distillate, allantoin and bisabalol).

It will be appreciated that a wide variety of different personal carecompositions can be prepared utilizing the above described ingredientsand other ingredients known in the art or which will be developed. It iswithin the skill in the art to chose appropriate ingredients andcombinations of ingredients and to determine an effective amount of theEPAC(s) of the invention to include in a particular personal carecomposition.

The invention also provides personal care devices. Personal care devicesinclude surgical materials (such as sutures and sponges), bandages,sponges, cloths, swabs, pads and wipes. The personal care devices of theinvention will have an EPAC or EPACs adhered to, absorbed into, adsorbedonto, bound to, attached to, entrapped in, impregnated in, coated ontoor otherwise incorporated into, them. For instance, a device can besoaked in a solution of an EPAC or EPACs, followed by removal of thesolvent, to adhere, absorb, adsorb, bind, attach, entrap, impregnate,coat the device with the EPAC(s). See, e.g., the description above ofthe preparation of oral care devices.

The invention also provides a method of inhibiting increasedphosphorylation in an animal's skin. The method comprises contacting theskin with an effective amount of an EPAC or EPACs. For instance, theskin may be contacted with a personal care composition comprising theEPAC(s). Methods of contacting the skin with personal care compositionsare well known in the art. Suitable methods include washing the skinwith a cleaning solution, rinsing the skin with a rinse, applying asolution, gel, cream, lotion or ointment on the skin (with or withoutthe use of an applicator), washing the hair with a shampoo that contactsthe scalp, and many other means of topical application. Suitableapplicators for applying personal care compositions include a cottonball, a gauze pad, a wipe, a cloth, a swab, a dropper, a syringe or afinger. In addition, the skin may be contacted with a personal caredevice comprising the EPAC(s). Methods of contacting the skin withpersonal care devices are well known in the art. For instance, suturescan be used to close a surgical wound, a wipe or pad impregnated withthe EPAC(s) can be used to clean the skin, a bandage comprising theEPACs can be applied to the skin, etc.

The treatment of the skin can be prophylactic treatment. For instance,the skin may be treated as part of a prophylactic skin care regimen. TheEPAC(s) can be incorporated into a personal care composition or devicethat is employed in such a regimen or the EPAC(s) may be contained in aseparate personal care composition or device which will be usedseparately from other compositions and devices employed in theprophylactic skin care regimen. The prophylactic regimen is performedregularly (e.g., monthly or daily).

Skin may also be treated prophylactically in connection with a varietyof dermatological procedures, including surgeries, dermabrasions andchemical peels. For instance, the area of skin on which surgery is to beperformed can be treated prior to surgery, during surgery, after thesurgery, or combinations thereof. For instance, the skin could be rinsedprior to surgery with a solution comprising the EPAC(s), the wound(s)caused by the surgery could be closed with sutures having the EPAC(s)incorporated into them, and/or the skin could be rinsed immediatelyafter the surgery, and/or at intervals thereafter, with a solutioncomprising the EPAC(s).

A personal care product comprising an EPAC or EPACs of the invention canalso be used to treat a disease or condition of the skin that ismediated by increased phosphorylation. Specific diseases and conditionstreatable according to the invention are described above in thediscussion of therapeutic methods and pharmaceutical compositions. Itwill be appreciated that diseases and conditions of the skin can betreated with a pharmaceutical composition and/or a personal carecomposition or device.

It is understood by those skilled in the art that the dosage amount ofthe EPAC(s) needed to treat an animal's skin using a personal careproduct will vary with the particular EPAC employed, whether thetreatment is prophylactic or for the treatment of a disease orcondition, the identity of the disease or condition to be treated, theseverity of the disease or condition, the type of personal carecomposition or device used, the duration of the treatment, the identifyof any other drugs being administered to the animal, the age, size andspecies of the animal, and like factors.

The invention also provides a kit comprising a personal care productaccording to the invention. In the case where the personal care productis a personal care composition, the kit may also include an applicatorfor applying the personal care composition, such as a swab, cottonballs, wipes, pads, a plastic paddle, a squeeze bottle, a pump bottle, adropper, or a syringe. The kit could also include a cup, vial or otherdevice for dispensing and/or measuring the amount of the personal carecomposition of the invention needed for the intended use. Of course, thekits could include both a personal care composition and a personal caredevice according to the invention. In addition to a personal carecomposition and/or device of the invention, the kits could also compriseanother type of personal care composition or device. Kits according tothe invention will also include instructions for using the kit and/orthe personal care product of the invention and may include any otherdesired items.

It is to be noted that “a” or “an” entity refers to one or more of thatentity. For example, “a tissue” refers to one or more tissues.

EXAMPLES Example 1 Inhibition of IL-8 By Dephosphorylated Phosvitin

Phosvitin was extracted from the yolks of chicken eggs as described byLosso and Nakai, in Egg Uses And Processing Technologies: NewDevelopments, pages 150-157 (Sim and Nakai, eds., Cab International,Oxon, UK, 1994). It was dephosphorylated as described by Reimerdes andKlostermeyer, in Methods in Enzymology, Volume 45, pages 26-28 (1976).This phosvitin preparation was estimated to be about 20-50%dephosphorylated.

Chicken phosvitin was also purchased from Sigma Chemical Co., St. Louis,Mo., as was bovine serum albumin (BSA; BUS grade=low fatty acid andIgO). Each was used as received from the manufacturer withoutdephosphorylation.

Jurkat cells (American Type Culture Collection (ATCC), Rockville, Md.)were cultured (1×10⁶ cells per 0.5 ml culture) in IMDM culture medium(ATCC) containing insulin transferrin selenite solution (ITSS; Sigma)with and without phosvitin or BSA at 37° C., 5% CO₂, for 30 minutes.Then, phorbol-12-myristate-13-acetate (PMA; 100 mg/ml; Sigma) andionomycin (100 mg/ml; Sigma) were added to stimulate the cells, and thecells were further cultured at 37° C., 5% CO₂, for 24 hours.

Cell culture supernatants were analyzed for IL-8 content by ELISA. TheIL-8 ELISA was performed as follows. Anti-human IL-8 antibody (PierceEndogen, Rockford, Ill.; catalogue number M801-E, lot number CK41959)was diluted to 1 μg/ml in phosphate buffered saline, pH 7.2-7.4, and 100μl of the diluted antibody was added to each well of Nunc Maxisorb ELISAstrip plates. The plates were incubated overnight at room temperature.The liquid was aspirated from the wells, and the plates were blotted ona paper towel. Then, 200 μl of assay buffer (phosphate buffered saline,pH 7.2-7.4, containing 4% BSA) were added to each well, and the plateswere incubated for 1 hour at room temperature. The liquid was aspiratedfrom the wells, and the wells were washed 3 times with wash buffer (50mM Tris, 0.2% Tween-20, pH 7.9-8.1) and were then blotted on a papertowel. Standards and supernatant samples (50 μl/well; standards werediluted in assay buffer) were added to the wells, and the plates wereincubated for 1 hour at room temperature with gentle shaking. The liquidwas aspirated, the wells were washed 3 times with wash buffer, and theplates were then blotted on a paper towel. Then, 100 μl ofbiotin-labeled anti-human IL-8 (Pierce Endogen, Rockford, Ill.;catalogue number M802-E, lot number CE49513), diluted to 60 ng/ml inassay buffer, were added to each well. The plates were incubated for 1hour at room temperature, the liquid was aspirated, the wells werewashed 3 times with wash buffer, and the plates were blotted on a papertowel. Then, 100 μl of HRP-conjugated streptavidin (Pierce Endogen,Rockford, Ill.; catalogue number N100) in assay buffer, were added toeach well. The plates were incubated for 30 minutes at room temperature,the liquid was aspirated, the wells were washed 3 times with washbuffer, and the plates were blotted on a paper towel. Finally, 100 μl ofTMB substrate solution (Pierce Endogen, Rockford, Ill.; catalogue numberN301) were added to each well. The plates were incubated for 30 minutesat room temperature. The reaction was stopped by adding 100 μl/well of0.18 M H₂SO₄. The optical densities at 450 mu and 530 nm were read on anELISA plate reader and the difference (OD 450-OD 530) calculated.

The results are shown in Table 1. As can be seen, it was found that thephosvitin isolated from chicken egg yolks inhibited IL-8 release ascompared to the positive control in a dose-dependent fashion. The BSAand phosvitin from Sigma did not significantly inhibit the release ofIL-8.

TABLE 1 Percent Inhibition Sample IL-8 (pg/ml) Of IL-8 Release Negativecontrol (no PMA, 0.0 — no ionomycin, no BSA, no phosvitin) Positivecontrol (PMA and 1119.2 — ionomycin; no BSA or phosvitin) 0.1 mg/mlchicken egg 1024.7  6.4% yolk phosvitin (dephosphorylated) and PMA andionomycin 0.5 mg/ml chicken egg 830.4 25.8% yolk phosvitin(dephosphoryalted) and PMA and ionomycin 1.0 mg/ml chicken egg 686.938.6% yolk phosvitin (dephosphorylated) and PMA and ionomycin 1.0 mg/mlSigma phosvitin 1153.6   0% (untreated) and PMA and ionomycin 1.0 mg/mlSigma BSA 1048.1  6.3% (untreated) and PMA and ionomycin

Example 2 Use of Dephosphorylated Phosvitin to Treat Inflammation

Chicken phosvitin (Sigma) in distilled water (0.5 g/100 ml) wasdephosphorylated by adding 0.4 N NaOH at 37° C. for 3 hours as describedin Jiang et al., J. Agric. Food Chem., 48:990-994 (2000). Afterdephosphorylation, the pH of the phosvitin solution was adjusted to 7.8with 0.1 N HCl. This phosvitin preparation was found to be 73%dephosphorylated using a gallium column.

A cream was prepared by mixing the dephosphorylated phosvitin with 100ml Eucerin cream (Beiersdorf Inc., Wilton, Conn.) to give a finalphosvitin concentration of about 0.25%.

A human volunteer with a chronic skin lesion (etiology unknown) appliedthe phosvitin cream to the lesion twice per day for two days. The lesiondisappeared completely after this treatment.

Another human volunteer with chronic skin lesions (diagnosed as atopicdermatitis) applied the phosvitin cream to a lesion on the back twiceper day for one week. The treated lesion completely disappeared. Otherlesions on this same volunteer that were not treated with the phosvitincream were unchanged.

A third human volunteer with a chronic skin lesion (diagnosed as chroniceczema) applied the phosvitin cream to the lesion twice per day for oneweek. The lesion completely disappeared after the treatment. Thisvolunteer's lesion has been resistant to all previous prescriptiontreatments, including topical steroids.

A fourth human volunteer with a chronic skin lesion on the top of thehead (diagnosed by biopsy to be chronic solar keratosis/elastosis)applied the phosvitin cream to the lesion twice per day for one week.The lesion almost completely disappeared. This volunteer's lesion hasbeen chronic for over 25 years and had not responded previously to anydermatological treatment, including topical steroids, antibiotics,antifungals, silver nitrate and freezing with liquid nitrogen.

A fifth human volunteer with a psoriasis lesion on the ankle applied thephosvitin cream to the lesion twice per day for one week. The lesioncompletely disappeared after this treatment.

Example 3 Inhibition of IL-8 by Unphosphorylated Kinase Substrates

Casein kinase I substrate (sequence Arg Arg Lys Asp Leu H is Asp Asp GluGlu Asp Glu Ala Met Ser Ile Thr Ala [SEQ ID NO:3]) and casein kinase IIsubstrate (sequence Arg Arg Arg Ala Asp Asp Ser Asp [SEQ ID NO:4]) werepurchased from Sigma-Aldrich, St. Louis, Mo. Each was used as receivedfrom the manufacturer since they were unphosphorylated.

Jurkat cells (American Type Culture Collection (ATCC), Rockville, Md.)were cultured (2×10⁶ cells per 1.0 ml culture) in IMDM culture medium(ATCC) containing insulin transferrin selenite solution (ITSS; Sigma)with and without each of the casein kinase substrates (50 μg/ml and 100μg/ml) at 37° C., 10% CO₂, for 15 minutes. Then,phorbol-12-myristate-13-acetate (PMA; 100 mg/ml; Sigma) and ionomycin(100 mg/ml; Sigma) were added to stimulate the cells, and the cells werefurther cultured at 37° C., 5% CO₂, for 24 hours.

Cell culture supernatants were analyzed for IL-8 content by ELISA asdescribed in Example 1. The results are shown in FIG. 1. As can be seen,it was found that the both of the casein kinase substrates inhibitedIL-8 release as compared to the positive control in a dose-dependentfashion.

1.-182. (canceled)
 183. A pharmaceutical composition comprising aphosphate acceptor compound (PAC) and a pharmaceutically-acceptablecarrier, wherein the PAC is a casein or a fragment thereof which is atleast partially dephosphorylated.
 184. The composition of claim 183wherein the casein is an α-casein, a β-casein, a γ-casein, a κ-casein, afragment of one of the foregoing, or a combination of one or more of theforegoing.
 185. The composition of claim 184 wherein the casein is anα_(S1)-casein or a fragment thereof.
 186. The composition of any one ofclaims 183-185 wherein the composition is formulated for topicaladministration.
 187. The composition of claim 186 which is drops, aspray, an aerosol or an inhalant.
 188. The composition of claim 186which is a powder or a foam.
 189. The composition of claim 186 which isa gel or a paste.
 190. The composition of claim 186 which is an ointmentor a cream.
 191. The composition of claim 186 which is a lotion. 192.The composition of claim 186 which is a wash, a rinse or a solution.